Hepatoweb Edition Mobile Téléchargement sur votre Agenda de Poche Cliquez ici

Mois de d'aout 2005

Archives depuis le 01/09/00

---

HEPATOLOGY

Table of Contents for August 2005 • Volume 42, Issue 2

Rapid Communication

Treatment of advanced hepatitis C with a low accelerating dosage regimen of antiviral therapy (p 255-262)
Gregory T. Everson, James Trotter, Lisa Forman, Marcelo Kugelmas, Arthur Halprin, Barbara Fey, Catherine Ray
Patients with advanced hepatitis C virus (HCV) are at risk of death and are candidates for liver transplantation. After transplantation, HCV recurs and may rapidly progress to cirrhosis and graft loss. Treatment is needed to prevent progression of disease and minimize recurrence after liver transplantation. We evaluated the effectiveness, tolerability, and outcome of a low accelerating dose regimen (LADR) of antiviral therapy in the treatment of patients with advanced HCV. One hundred twenty-four patients (male/female ratio 81:43; age range 37-71 years; 70% genotype 1) were treated with LADR. Sixty-three percent had clinical complications of cirrhosis (ascites, spontaneous bacterial peritonitis, varices, variceal hemorrhage, encephalopathy). The mean Child-Turcotte-Pugh (CTP) score was 7.4 ± 2.3, and the mean MELD score was 11.0 ± 3.7. Fifty-six patients were CTP class A, 45 were class B, and 23 were class C. Forty-six percent were HCV RNA-negative at end of treatment, and 24% were HCV RNA-negative at last follow-up. Sustained virological response (SVR) was 13% in patients infected with genotype 1 HCV and 50% in patients infected with non-1 genotypes (P < .0001). Non-1 genotype, CTP class A (genotype 1 only), and ability to tolerate full dose and duration of treatment (P < .0001) were predictors of SVR. Twelve of 15 patients who were HCV RNA-negative before transplantation remained HCV RNA-negative 6 months or more after transplantation. In conclusion, in a sizeable proportion of patients with advanced HCV, LADR may render blood free of HCV RNA, stabilize clinical course, and prevent posttransplantation recurrence.

Viral Hepatitis

Intrahepatic gene expression profiles and alpha-smooth muscle actin patterns in hepatitis C virus induced fibrosis (p 273-281)
Daryl T.-Y. Lau, Bruce A. Luxon, Shu-Yuan Xiao, Michael R. Beard, Stanley M. Lemon
To gain insight into pathogenic mechanisms underlying fibrosis in hepatitis C virus (HCV)-mediated liver injury, we compared intrahepatic gene expression profiles in HCV-infected patients at different stages of fibrosis and -smooth muscle actin (-SMA) staining patterns. We studied 21 liver biopsy specimens: 5 had no fibrosis (Ludwig-Batts stage 0); 10 had early portal or periportal fibrosis (stages 1 and 2); and 6, advanced fibrosis (stages 3 and 4). None of the patients had hepatocellular carcinoma. Transcriptional profiles were determined by high-density oligonucleotide microarrays. ANOVA identified 157 genes for which transcript abundance was associated with fibrosis stage. These defined three distinct hierarchical clusters of patients. Patients with predominantly stage 0 fibrosis had increased abundance of mRNAs linked to glycolipid metabolism. PDGF, a potent stellate cell mitogen, was also increased. Transcripts with increased abundance in stages 1 and 2 fibrosis were associated with oxidative stress, apoptosis, inflammation, proliferation, and matrix degradation, whereas transcripts increased in stages 3 and 4 were associated with fibrogenesis and cellular proliferation. Cells staining for -SMA were detectable at all stages but infrequent in advanced fibrosis without active inflammation. A high frequency of such cells was associated with mRNAs linked to glycolipid metabolism. In conclusion, the presence of -SMA-positive HSCs and expression of PDGF in stage 0 fibrosis suggests that stellate cells are activated early in HCV-mediated injury, possibly in response to oxidative stress resulting from inflammation and lipid metabolism. Increased abundance of transcripts linked to cellular proliferation in advanced fibrosis is consistent with a predisposition to cancer.

Predicting cirrhosis in patients with hepatitis C based on standard laboratory tests: Results of the HALT-C cohort (p 282-292)
Anna S. F. Lok, Marc G. Ghany, Zachary D. Goodman, Elizabeth C. Wright, Gregory T. Everson, Richard K. Sterling, James E. Everhart, Karen L. Lindsay, Herbert L. Bonkovsky, Adrian M. Di Bisceglie, William M. Lee, Timothy R. Morgan, Jules L. Dienstag, Chihiro Morishima, HALT-C Trial Group
Knowledge of the presence of cirrhosis is important for the management of patients with chronic hepatitis C (CHC). Most models for predicting cirrhosis were derived from small numbers of patients and included subjective variables or laboratory tests that are not readily available. The aim of this study was to develop a predictive model of cirrhosis in patients with CHC based on standard laboratory tests. Data from 1,141 CHC patients including 429 with cirrhosis were analyzed. All biopsies were read by a panel of pathologists (blinded to clinical features), and fibrosis stage was determined by consensus. The cohort was divided into a training set (n = 783) and a validation set (n = 358). Variables that were significantly different between patients with and without cirrhosis in univariate analysis were entered into logistic regression models, and the performance of each model was compared. The area under the receiver-operating characteristic curve of the final model comprising platelet count, AST/ALT ratio, and INR in the training and validation sets was 0.78 and 0.81, respectively. A cutoff of less than 0.2 to exclude cirrhosis would misclassify only 7.8% of patients with cirrhosis, while a cutoff of greater than 0.5 to confirm cirrhosis would misclassify 14.8% of patients without cirrhosis. The model performed equally well in fragmented and nonfragmented biopsies and in biopsies of varying lengths. Use of this model might obviate the requirement for a liver biopsy in 50% of patients with CHC. In conclusion, a model based on standard laboratory test results can be used to predict histological cirrhosis with a high degree of accuracy in 50% of patients with CHC.

Conventional liver CD4 T cells are functionally distinct and suppressed by environmental factors (p 293-300)
Steven C. Katz, Venu G. Pillarisetty, Joshua I. Bleier, T. Peter Kingham, Umer I. Chaudhry, Alaap B. Shah, Ronald P. DeMatteo
The contribution of intrahepatic conventional T cells to the unique immunologic properties of the liver has not been clearly defined. We isolated bulk and CD4 T cells from mouse liver and compared their functions with each other and with their splenic counterparts. Unlike bulk spleen T cells, bulk liver T cells reacted minimally to allogeneic or antigen-loaded syngeneic dendritic cells. However, after exclusion of natural killer T cells (NKTs) and T cells by FACS, liver and spleen CD4 T cells actually proliferated to a similar extent upon allogeneic or antigen-specific stimulation. Liver CD4 T cells were more sensitive to interleukin 2 (IL-2) than were spleen CD4 T cells, but had a similar proliferative potential based on their response to CD3 ligation. In addition, activated liver CD4 T cells produced higher levels of IL-4, IL-5, IL-10, and interferon gamma (IFN-) than did splenic CD4 T cells. Therefore, liver CD4 T cells are intrinsically different from spleen CD4 T cells. In vitro, liver or spleen NKTs and T cells suppressed liver and spleen CD4 T-cell proliferation in a dose-dependent fashion. In conclusion, unconventional T cells constrain liver CD4 T-cell function. Our findings have implications for pathological conditions of the liver that involve the response of conventional CD4 T lymphocytes.

APOBEC-mediated interference with hepadnavirus production (p 301-309)
Christine Rösler, Josef Köck, Michael Kann, Michael H. Malim, Hubert E. Blum, Thomas F. Baumert, Fritz von Weizsäcker
APOBEC3G is a cellular cytidine deaminase displaying broad antiretroviral activity. Recently, it was shown that APOBEC3G can also suppress hepatitis B virus (HBV) production in human hepatoma cells. In the present study, we characterized the mechanisms of APOBEC-mediated antiviral activity against HBV and related hepadnaviruses. We show that human APOBEC3G blocks HBV production in mammalian and nonmammalian cells and is active against duck HBV as well. Early steps of viral morphogenesis, including RNA and protein synthesis, binding of pregenomic RNA to core protein, and self-assembly of viral core protein, were unaffected. However, APOBEC3G rendered HBV core protein-associated full-length pregenomic RNA nuclease-sensitive. Ongoing reverse-transcription in capsids that had escaped the block in morphogenesis was not significantly inhibited. The antiviral effect was not modulated by abrogating or enhancing expression of the accessory HBV X protein, suggesting that HBV X protein does not represent a functional homologue to the HIV vif protein. Furthermore, human APOBEC3F but not rat APOBEC1 inhibited HBV DNA production. Viral RNA and low-level DNA produced in the presence of APOBEC3F or rat APOBEC1 occasionally displayed mutations, but the majority of clones were wild-type. In conclusion, APOBEC3G and APOBEC3F but not rat APOBEC1 can downregulate the production of replication-competent hepadnaviral nucleocapsids. In contrast to HIV and other retroviruses, however, APOBEC3G/3F-mediated editing of nucleic acids does not seem to represent an effective innate defense mechanism for hepadnaviruses.

Hepatic steatosis in HIV/hepatitis C coinfection: Prevalence and significance compared with hepatitis C monoinfection (p 310-316)
Alexander Monto, Lorna M. Dove, Alan Bostrom, Sanjay Kakar, Phyllis C. Tien, Teresa L. Wright
Liver disease in patients coinfected with HIV and hepatitis C virus (HCV) has received increasing attention in recent years. Steatosis is accepted as an important contributor to liver disease in patients with HCV, but despite coinfected patients having several reasons to have steatosis, the prevalence and significance of such changes has received scant attention. We examined steatosis in an unselected cohort of coinfected patients and compared its prevalence and predictors with findings in monoinfected patients, where these relationships have been established. We studied 92 coinfected and 372 monoinfected patients undergoing staging liver biopsy. Baseline characteristics of the two groups differed significantly, pointing at different contributors to steatosis in each. Histological inflammation and fibrosis were very similar in the two groups, but steatosis was less in coinfected patients. Steatosis had a univariate association with fibrosis in both groups, but retained a multivariate association only in monoinfected patients. Other multivariate predictors of steatosis in monoinfected patients were the accepted variables of elevated body mass index, male sex, and genotype 3a infection, as well as age. In coinfected patients, however, age was the only multivariate predictor. Undetectable HIV viral load was associated with steatosis in coinfected patients in univariate analysis, but highly active antiretroviral therapy or its individual components could not be initially linked to steatosis. In conclusion, steatosis is less common in HIV/HCV coinfected patients than similar HCV monoinfected patients, and predictors of steatosis differ between the two groups.

Liver Biology and Pathobiology

The modular microarchitecture of human liver (p 317-325)
Harald F. Teutsch
The morphological homogeneity of the liver parenchyma has represented a major obstacle in finding an acceptable definition of the structural/functional units of the liver. Concepts such as the lobule, the portal unit and the acinus remain debatable. This study investigates the modular microarchitecture on the basis of the lobular concept. Using alkaline phosphatase activity as a histochemical marker, modules could be recognized clearly. In autopsy specimens of human liver, modules were traced through sequential cryosections, and a secondary module having a height of 1.9 mm, a surface of 14.7 mm2, and a volume of 5.1 mm3 was reconstructed three-dimensionally. It was subdivided into 14 primary modules by portal tracts and vascular septa and by a common draining central venular tree. Primary modules were polyhedral, with seven to nine facets, having heights from 0.3 to 0.9 mm, surface areas from 1.7 to 5.0 mm2, and volumes from 0.1 to 0.9 mm3. Such variation in shape and size is considered an important part of the modular organization of the human liver. In conclusion, the findings on the three-dimensionality and microcirculation of liver modules support and extend the lobular concept and, at the same time, make apparent the shortcomings of the concepts of acinar and portal units. The results of this study should permit a better interpretation of histological sections of normal and pathological liver and provide a basis for understanding the metabolic heterogeneity of liver cells and their functional integration into parenchymal units.

The role of nuclear factor B in late-gestation liver development in the rat (p 326-334)
Michelle Embree-Ku, Philip A. Gruppuso
During the last 3 days of fetal development in the rodent, a burst of hepatocyte proliferation results in a tripling of liver size. Despite stimulation of mitogenesis via multiple signaling pathways, including some that are considered stress response pathways, little apoptosis accompanies this cell growth. Given the accepted role of nuclear factor B (NF-B) in preventing hepatocellular apoptosis during proliferation in mid-development, we predicted that NF-B would be functional during the period of rapid growth during late gestation in the rat. NF-B binding in electrophoretic mobility shift assays was low in embryonic day (E) 19 liver nuclear extracts relative to adult liver nuclear extracts. An additional band that was present in E19 liver was purified and identified as nucleolin. Tumor necrosis factor (TNF-) administration to E19 embryos in utero produced minimal induction of NF-B p50 homodimers and p50/p65 heterodimers, yet baseline apoptosis was not affected. Although p65 was present in E19 hepatocyte cytoplasm in amounts comparable to adult liver, we observed little translocation of p65 to the liver nuclei following TNF- administration. Additionally, expression of several NF-B-responsive genes remained minimally induced in E19 liver following TNF- treatment. In conclusion, although the NF-B components are present in late-gestation fetal liver, NF-B as a transcription factor is relatively inactive and unresponsive to TNF-. Given this finding and the high level of proliferation in late-gestation fetal liver, we predict that alternative antiapoptotic mechanisms are active during this period of rapid hepatic growth.

rAAV-mediated stable expression of heme oxygenase-1 in stellate cells: A new approach to attenuate liver fibrosis in rats (p 335-342)
Tung-Yu Tsui, Chi-Keung Lau, Jian Ma, Xiaobing Wu, Yan-Qing Wang, Stefan Farkas, Ruian Xu, Hans J. Schlitt, Sheung-Tat Fan
Liver fibrosis is the consequence of activation of hepatic stellate cells mediated by persistent or recurrent liver injury, where oxidative stress or inflammatory response resulting from immune cells and cytokines are involved. Targeting of hepatic stellate cells could be an important strategy for the therapy of liver fibrosis. In this study, we showed a tropism of recombinant adeno-associated virus (rAAV, serotype 2) with high efficiency in transduction of a homeostatic gene, heme oxygenase-1 (HO-1), to activated stellate cells. The binding of rAAVs to stellate cells increased significantly after serum-stimulated activation compared with quiescent status. Portal injection of rAAVs to normal or carbon tetrachloride (CCl4)-induced liver fibrosis showed a distinct distribution of rAAV binding. The majority of injected rAAVs bound to the cells in fibrotic areas that were associated with higher expression levels of fibroblast growth factor receptor-1 at 2 hours after administration. Isolation of different types of cells from CCl4-induced fibrotic livers showed predominant expression of transgene in stellate cells after rAAV/HO-1 administration on day 3 and remained stable for 12 weeks. In addition, HO-1-transduced stellate cells showed reduced transcript levels of type 1 collagen and impaired proliferative ability compared with controls. With this approach, the severity of established micronodular cirrhosis was markedly reduced. In conclusion, these findings suggest a new approach for the treatment of liver fibrosis using adeno-associated virus-mediated gene transfer.

Early response of 2(I) collagen to acetaldehyde in human hepatic stellate cells is TGF- independent (p 343-352)
Gianluca Svegliati-Baroni, Yutaka Inagaki, Ana-Rosa Rincon-Sanchez, Cindy Else, Stefania Saccomanno, Antonio Benedetti, Francesco Ramirez, Marcos Rojkind
Acetaldehyde is fibrogenic and induces the expression of type I collagen genes in hepatic stellate cells. Some of these acetaldehyde-dependent events are mediated by H2O2 and thus establish a direct connection between oxidative stress and collagen upregulation. We localized to the -378 to -183 region of the 2(I) collagen (COL1A2) promoter an acetaldehyde-responsive element (AcRE) functional in human hepatic stellate cells (HHSCs) and investigated molecular mechanisms whereby acetaldehyde stimulates and modulates its transcriptional activity. Because the AcRE co-localized with a previously described transforming growth factor (TGF-)1-responsive element, and both acetaldehyde and this cytokine induce their effects through H2O2, we investigated whether all fibrogenic actions of acetaldehyde were mediated by this cytokine. Here we show that acetaldehyde-induced COL1A2 upregulation in HHSCs recognizes two distinct but overlapping early and late stages that last from 1 to 6 hours and from 6 to 24 hours, respectively. We present several lines of evidence to show that early acetaldehyde-mediated events are independent of TGF-1. These include significant time-course differences in the expression of COL1A2 and TGF-1 mRNAs and inability of neutralizing antibodies to TGF-1 to inhibit acetaldehyde-dependent collagen gene transcription and Smad 3 phosphorylation. We also show that although acetaldehyde-dependent upregulation of collagen was PI3K dependent, that of TGF-1 was PI3K independent. In conclusion, acetaldehyde-dependent mechanisms involved in COL1A2 upregulation are similar, but not identical, to those of TGF-1. We suggest that early acetaldehyde-dependent events induce the late expression of TGF-1 and create an H2O2-dependent autocrine loop that may sustain and amplify the fibrogenic response of this alcohol metabolite.

Genotype-phenotype relationships in hepatocellular tumors from mice and man (p 353-361)
Sabine Stahl, Carina Ittrich, Philip Marx-Stoelting, Christoph Köhle, Özge Altug-Teber, Olaf Riess, Michael Bonin, Jürgen Jobst, Stephan Kaiser, Albrecht Buchmann, Michael Schwarz
Experimentally induced liver tumors in mice harbor activating mutations in either Catnb (-catenin) or Ha-ras, according to the carcinogenic treatment. We have now investigated by microarray analysis the gene expression profiles in tumors of the two genotypes. In total, 364 genes or expressed sequences with aberrant expression relative to normal liver were identified, but only 30 of these demonstrated unidirectional changes in both tumor types. Several functional clusters were identified that involve changes in amino acid utilization and ammonia disposition in Catnb-mutated tumors as opposed to alterations in lipid and cholesterol metabolism in Ha-ras-mutated tumors. Moreover, several genes coding for inhibitory molecules within the Wnt-signaling pathway were upregulated in Catnb-mutated tumors, suggesting induction of a negative feedback loop, whereas Ha-ras-mutated tumors showed alterations in the expression of several genes functional in monomeric G-protein signaling. We conclude that mouse hepatoma cells adopt different evolutionary strategies that allow for their selective outgrowth under variable environmental conditions. Human hepatocellular cancers (HCC) lack RAS mutations but are frequently mutated in CTNNB1, the human Catnb ortholog. The set of genes aberrantly expressed in Catnb-mutated mouse tumors was used to screen, by expression profiling, for dysregulation of orthologous genes within a panel of 25 HCCs, of which 10 were CTNNB1-mutated. HCCs with activated -catenin displayed a gene expression profile that was similar to Catnb-mutated mouse tumors but distinct from the other human HCCs. In conclusion, expression fingerprints may be used for diagnostic purposes and potential new therapeutic intervention strategies.

Antisense oligonucleotide reduction of DGAT2 expression improves hepatic steatosis and hyperlipidemia in obese mice (p 362-371)
Xing Xian Yu, Susan F. Murray, Sanjay K. Pandey, Sheri L. Booten, Dingjiu Bao, Xiu-Zhen Song, Susan Kelly, Songyuan Chen, Robert McKay, Brett P. Monia, Sanjay Bhanot
In this study, we investigated the role of acyl-coenzyme A:diacylglycerol acyltransferase 2 (DGAT2) in glucose and lipid metabolism in obese mice by reducing its expression in liver and fat with an optimized antisense oligonucleotide (ASO). High-fat diet-induced obese (DIO) C57BL/6J mice and ob/ob mice were treated with DGAT2 ASO, control ASO, or saline. DGAT2 ASO treatment reduced DGAT2 messenger RNA (mRNA) levels by more than 75% in both liver and fat but did not change DGAT1 mRNA levels in either of these tissues, which resulted in decreased DGAT activity in liver but not in fat. DGAT2 ASO treatment did not cause significant changes in body weight, adiposity, metabolic rate, insulin sensitivity, or skin microstructure. However, DGAT2 ASO treatment caused a marked reduction in hepatic triglyceride content and improved hepatic steatosis in both models, which was consistent with a dramatic decrease in triglyceride synthesis and an increase in fatty acid oxidation observed in primary mouse hepatocytes treated with DGAT2 ASO. In addition, the treatment lowered hepatic triglyceride secretion rate and plasma triglyceride levels, and improved plasma lipoprotein profile in DIO mice. The positive effects of the DGAT2 ASO were accompanied by a reduction in the mRNA levels of several hepatic lipogenic genes, including SCD1, FAS, ACC1, ACC2, ATP-citrate lyase, glycerol kinase, and HMG-CoA reductase. In conclusion, reduction of DGAT2 expression in obese animals can reduce hepatic lipogenesis and hepatic steatosis as well as attenuate hyperlipidemia, thereby leading to an improvement in metabolic syndrome.

CD154-CD40 interactions drive hepatocyte apoptosis in murine fulminant hepatitis (p 372-380)
Feng Zhou, Maureen N. Ajuebor, Paul L. Beck, Tai Le, Cory M. Hogaboam, Mark G. Swain
The CD154-CD40 interaction is a critical costimulatory pathway modulating the cellular immune response. Moreover, fulminant hepatitis of various etiologies is characterized by a hepatic influx of CD154-expressing T cells and an upregulation of CD40 expression on Kupffer cells and hepatocytes, implicating this pathway in the pathogenesis of fulminant hepatitis. In this study, we used a murine model of fulminant hepatitis induced by concanavalin A (con A) and documented a significant influx of CD154-expressing T cells into the livers of mice treated with con A, in association with markedly increased expression of CD40 restricted mainly to hepatocytes in damaged areas of the liver. Furthermore, con A hepatitis in CD154-deficient mice was significantly attenuated compared with that in wild-type controls and was associated with a decrease in hepatic tumor necrosis factor (TNF-) levels and hepatocyte death. We next determined the role of the CD154-CD40 pathway in hepatocyte death in vitro. These in vitro studies demonstrated that TNF- induces CD40 expression in hepatocytes and that subsequent activation of CD40 results in hepatocyte apoptosis mediated at least in part by enhanced hepatocyte expression of FasL. In conclusion, CD154 stimulation of CD40 plays a central role in hepatocyte death in fulminant hepatitis through direct and indirect pathways that may have direct therapeutic implications in humans.

Adenoviral gene transfer of ABIN-1 protects mice from TNF/galactosamine-induced acute liver failure and lethality (p 381-389)
Andy Wullaert, Ben Wielockx, Sofie Van Huffel, Veerle Bogaert, Bart De Geest, Peggy Papeleu, Peter Schotte, Karim El Bakkouri, Karen Heyninck, Claude Libert, Rudi Beyaert
Tumor necrosis factor (TNF) is a proinflammatory cytokine that plays a central role in acute and chronic hepatitis B and C infection and alcoholic liver disease as well as fulminant liver failure. TNF-induced liver failure is characterized by parenchymal cell apoptosis and inflammation leading to liver cell necrosis. The transcription factor NF-B is believed to mediate at least part of the proinflammatory effects of TNF, and is therefore a favorite drug target. However, NF-B also suppresses TNF-mediated hepatocyte apoptosis, implicating a potential cytotoxic effect of NF-B inhibitors in the liver. This dual function of NF-B emphasizes the need for therapeutics that can inhibit both TNF-induced NF-B activation and cell death. Here we describe that adenoviral expression of the NF-B inhibitory protein ABIN-1, but not an IB superrepressor (IBs), completely prevents lethality in the TNF/D-(+)-galactosamine-induced model of liver failure. Protection was associated with a significant decrease in TNF-induced leukocyte infiltration as well as hepatocyte apoptosis. The differential effects of ABIN-1 and IBs suggest a role for an NF-B independent function of ABIN-1. Indeed, ABIN-1 was found to prevent not only NF-B activation, but also apoptosis of cultured hepatocytes in response to TNF, explaining its protective effect against TNF-induced liver failure. In conclusion, ABIN-1 has a dual NF-B inhibitory and anti-apoptotic activity in the liver, which might be of considerable interest for the treatment of inflammatory liver diseases.

The role of hepatic type 1 plasminogen activator inhibitor (PAI-1) during murine hemorrhagic shock (p 390-399)
Claudio E. Lagoa, Yoram Vodovotz, Donna B. Stolz, Franck Lhuillier, Carol McCloskey, David Gallo, Runkuan Yang, Elena Ustinova, Mitchell P. Fink, Timothy R. Billiar, Wendy M. Mars
Hemorrhagic shock (HS) followed by resuscitation (HS-R) is characterized by profound physiological changes. Even if the patient survives the initial blood loss, these poorly understood changes can lead to morbidity. One of the tissues most often affected is liver. We sought to recognize specific hepatic changes induced by this stressor to identify targets for therapeutic intervention. Gene array analyses using mouse liver mRNAs were used to identify candidate genes that contribute to hepatic damage. To verify the role of one of the genes identified using the arrays, mice were subjected to HS-R, and multiple parameters were analyzed. A profound increase in plasminogen activator inhibitor type 1 (PAI-1) mRNA was observed using hepatic mRNAs from C57Bl/6 mice after HS, both with and without resuscitation. Constitutive loss of PAI-1 resulted in notable tissue preservation and lower (P < .05) alanine aminotransferase (ALT) levels. Fibrin degradation products (FDPs) and interleukins 6 and 10 (IL-6 and IL-10) were unaffected by loss of PAI-1; however, enhanced urokinase activity, an elevation of active hepatocyte growth factor (HGF), an increase in unprocessed transforming growth factor-1 (TGF-1), and retention of ERK phosphorylation after HS-R were associated with improved hepatic function. In conclusion, PAI-1 protein is a negative effector of hepatic damage after HS-R through its influence on classic regulators of hepatic growth, as opposed to its role in fibrinolysis.

Induction of cytochrome P450 2E1 increases hepatotoxicity caused by Fas agonistic Jo2 antibody in mice (p 400-410)
Xiaodong Wang, Yongke Lu, Arthur I. Cederbaum
Cytochrome P450 2E1 (CYP2E1) may be a central pathway in generating oxidative stress, reactive oxygen species, and causing hepatotoxic injury by alcohol and various hepatotoxins. This study evaluated the ability of CYP2E1 to potentiate or synergize the hepatotoxicity of Fas in vivo. C57BL/6 mice were injected intraperitoneally with pyrazole (Pyr) to induce CYP2E1. Then, 16-hour fasted mice were administered agonistic Jo2 anti-Fas antibody ip. Other mice were treated with Pyr or Jo2 alone. Levels of serum aminotransferase were 8.3- and 6.3-fold higher in the Pyr/Jo2 group compared with Jo2 alone, respectively. Histological evaluation of liver showed more extensive acidophilic necrosis and severe pathological changes in the Pyr/Jo2-treated mice. DNA fragmentation and caspase-8 and -3 activities were more elevated in the Pyr/Jo2 group compared with Jo2 alone. CYP2E1 activity and protein levels were higher in the Pyr/Jo2 group than in Jo2 alone. Levels of inducible nitric oxide synthase, 3-nitrotyrosine protein adducts, malondialdehyde, and protein carbonyls were also higher in the Pyr/Jo2 group compared with Jo2 alone. Glutathione and activities of catalase and Cu-Zn superoxide dismutase were decreased in the Pyr/Jo2 group. Administration of chlormethiazole, an inhibitor of CYP2E1, to the Pyr/Jo2-treated mice caused a significant decrease of alanine aminotransferase and liver pathological changes in association with a decrease in CYP2E1 protein and activity. In conclusion, enhanced hepatotoxicity of Fas was found in mice with elevated levels of CYP2E1. We speculate that overexpression of CYP2E1 might synergize and increase the susceptibility to Fas induced-liver injury.

Mesenteric Th1 polarization and monocyte TNF- production: First steps to systemic inflammation in rats with cirrhosis (p 411-419)
Leticia Muñoz, Agustín Albillos, Mónica Nieto, Eduardo Reyes, Lourdes Lledó, Jorge Monserrat, Eva Sanz, Antonio de la Hera, Melchor Alvarez-Mon
A systemic inflammatory state with increased circulating tumor necrosis factor alpha (TNF-) has been related to the bacterial infection susceptibility and hemodynamic derangement of patients with cirrhosis. We compared the activation status of immune cell subpopulations defined by 4-color cytometry in mesenteric and peripheral lymph nodes and blood of rats with CCl4-cirrhosis to define the immune response initiation site, the T-cell and monocyte contribution to pro-inflammatory cytokine production, as well as the pathogenic role of enteric bacteria in the cirrhosis immune response. Th1 cells and monocytes were expanded in the mesenteric nodes (P < .001) and blood (P < .001) of rats with cirrhosis, and activated to produce interferon gamma (P < .0001) and TNF- (P < .0001), respectively. The greater numbers of recently activated CD134+ Th cells in mesenteric nodes compared with blood, the correlation between their numbers in mesenteric nodes and blood (r = 0.66, P < .001), and the expansion of activated CD45RC- Th cells, which are unable to re-enter lymph nodes, in mesenteric nodes but not in blood or axillary nodes points to mesenteric nodes as the origin site of activated Th cells. Abrogation of bacterial translocation by bowel decontamination reduced the number of activated Th cells and monocytes, and normalized interferon gamma production by Th cells and TNF- production by monocytes in mesenteric nodes and blood, respectively. In conclusion, in cirrhosis, enteric bacteria start off an orchestrated immune response cascade in mesenteric nodes involving Th1 polarization and monocyte activation to TNF- production. Later, the recirculation of these activated effector immune cells into blood promotes systemic inflammation.

CAR and PXR agonists stimulate hepatic bile acid and bilirubin detoxification and elimination pathways in mice (p 420-430)
Martin Wagner, Emina Halilbasic, Hanns-Ulrich Marschall, Gernot Zollner, Peter Fickert, Cord Langner, Kurt Zatloukal, Helmut Denk, Michael Trauner
Induction of hepatic phase I/II detoxification enzymes and alternative excretory pumps may limit hepatocellular accumulation of toxic biliary compounds in cholestasis. Because the nuclear xenobiotic receptors constitutive androstane receptor (CAR) and pregnane X receptor (PXR) regulate involved enzymes and transporters, we aimed to induce adaptive alternative pathways with different CAR and PXR agonists in vivo. Mice were treated with the CAR agonists phenobarbital and 1,4-bis-[2-(3,5-dichlorpyridyloxy)]benzene, as well as the PXR agonists atorvastatin and pregnenolone-16-carbonitrile. Hepatic bile acid and bilirubin-metabolizing/detoxifying enzymes (Cyp2b10, Cyp3a11, Ugt1a1, Sult2a1), their regulatory nuclear receptors (CAR, PXR, farnesoid X receptor), and bile acid/organic anion and lipid transporters (Ntcp, Oatp1,2,4, Bsep, Mrp2-4, Mdr2, Abcg5/8, Asbt) in the liver and kidney were analyzed via reverse-transcriptase polymerase chain reaction and Western blotting. Potential functional relevance was tested in common bile duct ligation (CBDL). CAR agonists induced Mrp2-4 and Oatp2; PXR agonists induced only Mrp3 and Oatp2. Both PXR and CAR agonists profoundly stimulated bile acid-hydroxylating/detoxifying enzymes Cyp3a11 and Cyp2b10. In addition, CAR agonists upregulated bile acid-sulfating Sult2a1 and bilirubin-glucuronidating Ugt1a1. These changes were accompanied by reduced serum levels of bilirubin and bile acids in healthy and CBDL mice and by increased levels of polyhydroxylated bile acids in serum and urine of cholestatic mice. Atorvastatin significantly increased Oatp2, Mdr2, and Asbt, while other transporters and enzymes were moderately affected. In conclusion, administration of specific CAR or PXR ligands results in coordinated stimulation of major hepatic bile acid/bilirubin metabolizing and detoxifying enzymes and hepatic key alternative efflux systems, effects that are predicted to counteract cholestasis.

Successful gene therapy of the Gunn rat by in vivo neonatal hepatic gene transfer using murine oncoretroviral vectors (p 431-438)
Marta Bellodi-Privato, Dominique Aubert, Virginie Pichard, Anne Myara, François Trivin, Nicolas Ferry
Crigler-Najjar type 1 disease (CN1) is a rare inherited metabolic disease characterized by complete absence of hepatic UDP-glucuronosyl transferase (UGT1), resulting in high levels of unconjugated bilirubin. CN1 is an attractive candidate disease for gene therapy. Here we show that in vivo neonatal hepatocyte transduction using recombinant oncoretroviral vectors results in long-term and complete phenotype correction in Gunn rats, a model for CN1. Two-day-old newborn Gunn rats were injected via the temporal vein with 200 L UGT1 or control -galactosidase retroviral vectors. In UGT1-injected animals, bilirubinemia was normal at 6 weeks (3 mol/L) and remained in the normal range (i.e., <10 mol/L) for more than 34 weeks. In contrast, in -galactosidase-injected animals as well as in noninjected controls, bilirubinemia remained at a high level (i.e., >100 mol/L) during the whole experimental follow-up. Large amounts of bilirubin monoglucuronides and diglucuronides were present in the bile of treated animals. Finally, polymerase chain reaction and reverse transcription polymerase chain reaction analysis as well as Western blot confirmed the presence and expression of UGT1 almost exclusively in the liver. The estimated proportion of transduced hepatocytes was in the range of 5% to 10%. In conclusion, complete and permanent correction of hyperbilirubinemia in newborn Gunn rats using retroviral vectors can be obtained, paving the way for future gene therapy for CN1.

Liver Failure and Liver Disease

Circulatory function and hepatorenal syndrome in cirrhosis (p 439-447)
Luis Ruiz-del-Arbol, Alberto Monescillo, Carlos Arocena, Paz Valer, Pere Ginès, Víctor Moreira, José María Milicua, Wladimiro Jiménez, Vicente Arroyo
The pathogenic mechanism of hepatorenal syndrome is not well established. We investigated the circulatory function in cirrhosis before and after the development of hepatorenal syndrome. Systemic and hepatic hemodynamics and the activity of endogenous vasoactive systems were measured in 66 patients who had cirrhosis with tense ascites and normal serum creatinine levels; measurements were repeated at follow-up in 27 cases in whom hepatorenal syndrome had developed. At baseline, mean arterial pressure and cardiac output were significantly higher, and hepatic venous pressure gradient, plasma renin activity, and norepinephrine concentration were significantly lower in patients who did not develop hepatorenal syndrome compared with those presenting with this complication. Peripheral vascular resistance was decreased to the same extent in the two groups. Plasma renin activity and cardiac output were the only independent predictors of hepatorenal syndrome. Hepatorenal syndrome occurred in the setting of a significant reduction in mean arterial pressure (83 ± 9 to 75 ± 7 mmHg; P < .001), cardiac output (6.0 ± 1.2 to 5.4 ± 1.5 L/min; P < .01), and wegded pulmonary pressure (9.2 ± 2.6 to 7.5 ± 2.6 mmHg; P < .001) and an increase in plasma renin activity (9.9 ± 5.2 to 17.5 ± 11.4 ng/mL · hr; P < .001), norepinephrine concentration (571 ± 241 to 965 ± 502 pg/mL; P < .001), and hepatic venous pressure gradient. No changes were observed in peripheral vascular resistance. In conclusion, these data indicate that hepatorenal syndrome is the result of a decrease in cardiac output in the setting of a severe arterial vasodilation.

UGT1A1 polymorphisms are important determinants of dietary carcinogen detoxification in the liver (p 448-457)
Hugo Girard, Jean Thibaudeau, Michael H. Court, Louis-Charles Fortier, Lyne Villeneuve, Patrick Caron, Qin Hao, Lisa L. von Moltke, David J. Greenblatt, Chantal Guillemette
PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-f]pyridine), the most abundant heterocyclic amine in diet, is involved in the etiology of cancer. PhIP and its carcinogenic metabolite N-hydroxy-PhIP (N-OH-PhIP) are extensively conjugated by UDP-glucuronosyltransferase (UGTs) with wide variability. This study aimed to determine the genetic influence of UGTs on the hepatic detoxification of this carcinogen. The formation of N-OH-PhIP glucuronides was studied in 48 human liver samples by mass spectrometry. Liver samples were genotyped for common polymorphisms and correlated with UGT protein levels and N-OH-PhIP glucuronidation activities. The formation of four different N-OH-PhIP glucuronide metabolites was observed in all livers. The major metabolite was N-OH-PhIP-N2-glucuronide (N2G), which is the primary metabolite found in human urine, and showed a high interindividual variability (up to 28-fold). Using an heterologous expression system, the bilirubin-conjugating UGT1A1 enzyme was identified among all known UGTs (n = 16) as the predominant enzyme involved. The significant correlation between UGT1A1 protein content and formation of N2G (Rs = 0.87; P < .0001) suggests a critical role for UGT1A1 in the hepatic metabolism of this carcinogen. UGT1A1 expression was strongly determined by the presence of the common promoter polymorphisms, UGT1A1*28 (TATA box polymorphism) (P = .0031), -3156G/A (P = .0006) and -3279G/T (P = .0017), and rates of N2G were indeed correlated with these polymorphisms (P < .05), whether analyzed individually or in combination (haplotypes). In conclusion, UGT1A1 polymorphisms modulate the hepatic metabolism of the carcinogenic intermediate of PhIP and may determine the level of its exposure and potentially influence the risk of cancer through dietary exposure to HCAs.

Primary biliary cirrhosis is characterized by IgG3 antibodies cross-reactive with the major mitochondrial autoepitope and its Lactobacillus mimic (p 458-465)
Dimitrios-Petrou Bogdanos, Harold Baum, Manabu Okamoto, Paolo Montalto, Umesh C. Sharma, Eirini I. Rigopoulou, John Vlachogiannakos, Yun Ma, Andrew K. Burroughs, Diego Vergani
The serological hallmark of primary biliary cirrhosis (PBC) is the presence of pyruvate dehydrogenase complex E2 subunit (PDC-E2) antimitochondrial antibodies (AMAs). Anti-PDC-E2 antibodies cross-react specifically with mycobacterial hsp65, and we have demonstrated that the motif SxGDL[ILV]AE shared by PDC-E2212-226 and hsp's is a cross-reactive target. Having found that this same motif is present only in -galactosidase of Lactobacillus delbrueckii (BGAL LACDE), we hypothesized that this homology would also lead to cross-reactivity. The mimics were tested via ELISA for reactivity and competitive cross-reactivity using sera from 100 AMA-positive and 23 AMA-negative PBC patients and 190 controls. An Escherichia coli (ECOLI) PDC-E2 mimic that has been pathogenetically linked to PBC but lacks this motif has been also tested. Anti-BGAL266-280 LACDE antibodies were restricted to AMA-positive patients (54 of 95, 57%) and belonged to immunoglobulin (Ig) G3. Of the 190 controls, 22 (12%; P < .001) had anti-BGAL266-280 antibodies, mainly of the IgG4 subclass. ECOLI PDC-E2 reactivity was virtually absent. BGAL266-280/PDC-E2212-226 reactivity of the IgG3 isotype was found in 52 (52%) AMA-positive PBC patients but in only 1 of the controls (P < .001). LACDE BGAL266-280/PDC-E2212-226 reactivity was due to cross-reactivity as confirmed via competition ELISA. Antibody affinity for BGAL266-280 was greater than for PDC-E2 mimics. Preincubation of a multireactive serum with BGAL266-280 reduced the inhibition of enzymatic activity by 40%, while marginal effect (12%) or no effect (2%) was observed in human or ECOLI PDC-E2 mimics. In conclusion, IgG3 antibodies to BGAL LACDE cross-react with the major mitochondrial autoepitope and are characteristic of PBC.

Primary iron overload with inappropriate hepcidin expression in V162del ferroportin disease (p 466-472)
Heinz Zoller, Ian McFarlane, Igor Theurl, Sylvia Stadlmann, Elizabeta Nemeth, David Oxley, Tomas Ganz, David J. Halsall, Timothy M. Cox, Wolfgang Vogel
Ferroportin disease (hemochromatosis type 4) is a recently recognized disorder of human iron metabolism, characterized by iron deposition in macrophages, including Kupffer cells. Mutations in the gene encoding ferroportin 1, a cellular iron exporter, are responsible for this iron storage disease, inherited as an autosomal dominant trait. We present clinical, histopathological, and radiological findings in a family with the most common ferroportin mutation, V162del. In the index case, the disorder is characterized by abundant deposition of hemosiderin in all tissues investigated (mesenteric lymph node, liver, gastric and duodenal mucosa, and also in squamous cell carcinoma of the lung). The radiological findings indicated the presence of excess iron in bone marrow and spleen. Despite a significant burden of iron, no features of chronic liver disease were found in affected members of the family, including individuals aged up to 80 years. Hyperferritinemia greater than 1,000 g/L was a penetrant biochemical finding before the second decade in life and was associated with significantly increased serum concentrations of pro-hepcidin that correlated positively with urinary hepcidin concentrations. In conclusion, the systemic iron burden in ferroportin disease is not a sufficient cause for chronic liver disease. In patients with most, but not all, ferroportin mutations, retention of iron in macrophages of the liver and other organs may protect against damage to parenchymal cells. Finally, macrophage iron storage in ferroportin disease is associated with elevated serum pro-hepcidin levels.

Endothelial dysfunction and cardiovascular risk profile in nonalcoholic fatty liver disease (p 473-480)
Nicola Villanova, Simona Moscatiello, Stefano Ramilli, Elisabetta Bugianesi, Donatella Magalotti, Ester Vanni, Marco Zoli, Giulio Marchesini
Nonalcoholic fatty liver disease (NAFLD) is consistently associated with features of the metabolic syndrome, a condition carrying a high risk of cardiovascular events. We measured the vasodilatory response of the brachial artery in response to ischemia (a test of endothelial function) (FMV) as well as cardiovascular risk profile in 52 NAFLD cases and 28 age- and sex-matched controls. The 10-year risk of coronary events was calculated according to the Framingham equation and the scores derived from the PROCAM study and NCEP-ATPIII proposals. FMV was 6.33% ± 5.93% in NAFLD versus 12.22% ± 5.05% in controls (P < .0001), and higher in pure fatty liver (9.93%) compared with nonalcoholic steatohepatitis (4.94%) (P = .010). No differences were observed in flow-independent vasodilation (response to sublingual nitroglycerin). Percent FMV was negatively associated with insulin resistance (homeostasis model assessment) in the whole population (r = -0.243; P = .030). In logistic regression analysis, NAFLD was associated with a percent FMV in the lower tertile (OR, 6.7; 95% CI, 1.26-36.1), after adjustment for age, sex, body mass index, and insulin resistance. Among NAFLD patients, low FMV was associated with nonalcoholic steatohepatitis (adjusted OR, 6.8; 95% CI, 1.2-40.2). The 10-year probability of cardiovascular events was moderately increased in NAFLD, and particularly in nonalcoholic steatohepatitis. In conclusion, our study provides evidence of endothelial dysfunction and increased risk of cardiovascular events in NAFLD. The risk of advanced liver disease is well recognized in NAFLD patients, but the large majority of cases might experience cardiovascular disease in the long term, indirectly limiting the burden of liver failure.

Outcome and prognostic markers in severe drug-induced liver disease (p 481-489)
Einar Björnsson, Rolf Olsson
The combination of high aminotransferases (hepatocellular injury) and jaundice has been reported to lead to a mortality rate of 10% to 50% for different drugs, a phenomenon known as Hy's rule. However, Hy's rule has never been validated, and limited data exist on predictors for outcome in hepatocellular and other forms of drug-induced liver disease. All reports of suspected hepatic adverse drug reactions received by the Swedish Adverse Drug Reactions Advisory Committee (1970-2004) were reviewed. Cases with bilirubin levels 2 or more times the upper limit of normal (ULN) were analyzed. A total of 784 cases were retrieved - 409 with hepatocellular injury, 206 with cholestatic injury, and 169 with mixed liver injury. The mortality/transplantation rate was 9.2%, and bilirubin (median 18.7 ? ULN [IQR 12.6-25]; range 4.5-42) was higher (P < .0001) in the deceased/transplant recipients compared with the surviving patients (median 5.5 ? ULN [IQR 3.3-9.5]; range 2.0-38). A total of 7.8% with cholestatic and 2.4% with a mixed pattern died. The mortality rate in hepatocellular injury for different drugs varied from 40% (6 of 15) for halothane to 0% (0 of 32) for erythromycin, in total 12.7%. Using logistic regression analysis, age, aspartate aminotransferase (AST) and bilirubin were found to independently predict death or liver transplantation in the hepatocellular group, whereas among patients with cholestatic/mixed liver injury, bilirubin was the only independent predictor. In conclusion, hepatocellular jaundice has a high but variable mortality rate, depending on the drug involved. The AST and bilirubin levels are the most important predictors of death or liver transplantation.

Copyright © 2005 by the American Association for the Study of Liver Diseases. All rights reserved.

GASTROENTEROLOGY

Rapid communication

Mannose Binding Lectin Gene Polymorphisms Confer a Major Risk for Severe Infections After Liver Transplantation
Bouwman LH, Roos A, Terpstra OT, de Knijff P, van Hoek B, Verspaget HW, Berger SP, Daha MR, Frölich M, van der Slik AR, Doxiadis II, Roep BO, Schaapherder AFM
Background & Aims: Infection is the primary cause of death after liver transplantation. Mannose binding lectin (MBL) is a recognition molecule of the lectin pathway of complement and a key component of innate immunity. MBL variant alleles have been described in the coding region of the MBL gene, which are associated with low MBL serum concentration and impaired MBL structure and function. The aims of our study were to establish the role of the liver in production of serum MBL and to evaluate the effect of MBL variant alleles on the susceptibility to infection after liver transplantation. Methods: We investigated 49 patients undergoing orthotopic liver transplantation. MBL exon 1 and promoter polymorphisms were determined in patients and in liver donors. MBL serum concentration was determined before and during 1 year after transplantation. The incidence of clinically significant infections during this period was assessed. Results: Transplantation of MBL wildtype recipients with donor livers carrying MBL variant alleles resulted in a rapid and pronounced decrease of serum MBL levels. This serum conversion was associated with the disappearance of high molecular weight MBL. No indication for extrahepatic production of serum MBL could be obtained. The presence of MBL variant alleles in the MBL gene of the donor liver, but not in the recipient, was associated with a strongly increased incidence of clinically significant infections after transplantation. Conclusions: Serum MBL is produced by the liver under strong genetic control. After liver transplantation, the MBL genotype of the donor liver is a major risk determinant for life-threatening infections.

Clinical-alimentary tract

Cancer Risk in Hereditary Nonpolyposis Colorectal Cancer Syndrome: Later Age of Onset
Hampel H, Stephens JA, Pukkala E, Sankila R, Aaltonen LA, Mecklin JP, de la Chapelle A
Background & Aims: Mutations in the mismatch repair genes cause hereditary nonpolyposis colorectal cancer (HNPCC) syndrome and convey high lifetime cancer risks for colorectal (CRC) and endometrial cancer. Currently, cancer risks for individuals with HNPCC are based on data from clinically ascertained families. The purpose of this study was to re-examine the penetrance in HNPCC using a comprehensive dataset from a geographically defined region. Methods: A combined dataset of 70 HNPCC families ascertained by traditional high-risk criteria and by molecular screening comprising 88 probands and 373 mutation-positive family members was used. Statistical methods were modified survival analysis techniques. Results: In mutation-positive relatives (excluding probands), the median age at diagnosis of CRC was 61.2 years (confidence interval [CI], 56.3–68.0 y). The lifetime risk for CRC was 68.7% (CI, 58.6%–78.9%) for men and 52.2% (CI, 37.6%–66.9%) for women. Considering only probands, the median age at diagnosis of CRC was 44.0 years (CI, 41.0–46.3 y). Median age of onset of EC was 62.0 years (CI, 55.9 y to an upper limit too high to calculate) with a lifetime cancer risk of 54% (CI, 41.9%–66.1%). Conclusions: A markedly later age of onset for CRC at 61 y than previously reported (?44 y) is suggested, resulting mainly from a more rigorous method of analysis in which all gene-positive individuals (both affected and unaffected with cancer) are considered. Lifetime cancer risks may be lower for CRC and endometrial cancer than presently assumed. If confirmed, these data suggest a need to alter counseling practices, and to consider HNPCC in older individuals than before.

A Comparison of the Immunochemical Fecal Occult Blood Test and Total Colonoscopy in the Asymptomatic Population
Morikawa T, Kato J, Yamaji Y, Wada R, Mitsushima T, Shiratori Y
Background & Aims: The fecal occult blood test (FOBT) is recommended as a screening test for colorectal cancer, but there are few reliable studies on the accuracy of immunochemical FOBT. The aim of this study was to analyze the sensitivity of immunochemical FOBT and to compare the results with the findings from complete colonoscopy. Methods: Asymptomatic adults underwent 1-time immunochemical FOBT and total colonoscopy simultaneously. The prevalence and location of colorectal neoplasia were determined by colonoscopy. The results of immunochemical FOBT and the colonoscopic findings were compared. Results: Of 21,805 patients, immunochemical FOBT was positive in 1231 cases (5.6%). The sensitivity of 1-time immunochemical FOBT for detecting advanced neoplasia and invasive cancer was 27.1% and 65.8%, respectively. In addition, the sensitivity for invasive cancer according to Dukes’ stage showed 50.0% for Dukes’ stage A, 70.0% for Dukes’ stage B, and 78.3% for Dukes’ stages C or D. The sensitivity for detecting advanced neoplasia at the proximal colon was significantly lower than that detected in the distal colon (16.3% vs 30.7%, P = .00007). Conclusions: Although the screening of asymptomatic patients with immunochemical FOBT can identify patients with colorectal neoplasia to a certain extent, the sensitivity is relatively low and different according to the tumor location. Therefore, programmatic and repeated screening by immunochemical FOBT may be necessary to increase sensitivity for colorectal cancer detection.

The Perception of Cancer Risk in Patients With Prevalent Barrett’s Esophagus Enrolled in an Endoscopic Surveillance Program
Shaheen NJ, Green B, Medapalli RK, Mitchell KL, Wei JT, Schmitz SM, West LM, Brown A, Noble M, Sultan S, Provenzale D
Background&Aims: Patients with Barrett’s esophagus (BE) have a risk of esophageal adenocarcinoma of approximately 0.5% per year. Patients may have difficulty understanding this risk. This study assessed the perceived risk of cancer in patients with BE, and correlated their risk estimates with their health care use behaviors. Methods: We performed a survey of patients with BE participating in an endoscopic surveillance program at 2 sites: a university teaching hospital and a Veterans’ Administration hospital. A questionnaire also elicited their demographics as well as their sources of health information. Health care behaviors, including physician visits and endoscopic surveillance behaviors, were assessed. Patients were classified as either overestimators or nonoverestimators of risk. Characteristics of overestimators, as well as health care use patterns, were assessed. Results: One hundred eighteen patients met entry criteria, and 92 (78%) completed all the questionnaires. Sixty-eight percent of patients overestimated their 1-year risk of cancer, with a mean estimated 1-year cancer risk being 13.6%. The lifetime risk also was overestimated by 38% of patients. Patients who overestimated risk were more likely to be Veterans’ Administration medical center patients, have more symptomatic reflux, and were more likely to use the Internet to get health care information. There was no significant difference in physician visits between overestimators and nonestimators (1.2 visits per year vs 1.0, P = .20), nor in endoscopy use (5.7 endoscopies per 5-year period vs 5.0, P = .42). Conclusions: The majority of patients with prevalent BE participating in an endoscopic surveillance program overestimated their chances of developing adenocarcinoma of the esophagus. Efforts to improve education of such patients with BE are warranted.

Differential Expression of Cannabinoid Receptors in the Human Colon: Cannabinoids Promote Epithelial Wound Healing
Wright K, Rooney N, Feeney M, Tate J, Robertson D, Welham M, Ward S
Background & Aims: Two G-protein—coupled cannabinoid receptors, termed CB1 and CB2, have been identified and several mammalian enteric nervous systems express CB1 receptors and produce endocannabinoids. An immunomodulatory role for the endocannabinoid system in gastrointestinal inflammatory disorders has been proposed and this study sought to determine the location of both cannabinoid receptors in human colon and to investigate epithelial receptor function. Methods: The location of CB1 and CB2 receptors in human colonic tissue was determined by immunohistochemistry. Primary colonic epithelial cells were treated with both synthetic and endogenous cannabinoids in vitro, and biochemical coupling of the receptors to known signaling events was determined by immunoblotting. Human colonic epithelial cell lines were used in cannabinoid-binding studies and as a model for in vitro wound-healing experiments. Results: CB1-receptor immunoreactivity was evident in normal colonic epithelium, smooth muscle, and the submucosal myenteric plexus. CB1- and CB2-receptor expression was present on plasma cells in the lamina propria, whereas only CB2 was present on macrophages. CB2 immunoreactivity was seen in the epithelium of colonic tissue characteristic of inflammatory bowel disease. Cannabinoids enhanced epithelial wound closure either alone or in combination with lysophosphatidic acid through a CB1—lysophosphatidic acid 1 heteromeric receptor complex. Conclusions: CB1 receptors are expressed in normal human colon and colonic epithelium is responsive biochemically and functionally to cannabinoids. Increased epithelial CB2-receptor expression in human inflammatory bowel disease tissue implies an immunomodulatory role that may impact on mucosal immunity.

Celiac Disease and Risk of Adverse Fetal Outcome: A Population-Based Cohort Study
Ludvigsson JF, Montgomery SM, Ekbom A
Background & Aims: Studies of maternal celiac disease (CD) and fetal outcome are inconsistent, and low statistical power is likely to have contributed to this inconsistency. We investigated the risk of adverse outcomes in women with CD diagnosed prior to pregnancy and in women who did not receive a diagnosis of CD until after the delivery. Methods: A national register-based cohort study restricted to women aged 15–44 years with singleton live born infants was used. We identified 2078 offspring to women who had received a diagnosis of CD (1964–2001): 1149 offspring to women diagnosed prior to birth and 929 offspring to women diagnosed after infant birth. Main outcome measures were: intrauterine growth retardation, low birth weight (<2500 g), very low birth weight (<1500 g), preterm birth (<37 gestational weeks), very preterm birth (<30 gestational weeks), and caesarean section. Results: Undiagnosed CD was associated with an increased risk of intrauterine growth retardation (OR = 1.62; 95% CI: 1.22–2.15), low birth weight (OR = 2.13; 95% CI: 1.66–2.75), very low birth weight (OR = 2.45; 95% CI: 1.35–4.43), preterm birth (OR = 1.71; 95% CI: 1.35–2.17), and caesarean section (OR = 1.82; 95% CI: 1.27–2.60). In contrast, a diagnosis of CD made before the birth was not associated with these adverse fetal outcomes. Conclusions: Undiagnosed maternal CD is a risk factor for unfavorable fetal outcomes, but the risks are reduced when CD has been diagnosed. CD diagnosed prior to pregnancy does not constitute a great a risk as undiagnosed CD.

Insulin-Like Growth Factor-I and Insulin Are Associated With the Presence and Advancement of Adenomatous Polyps
Schoen RE, Weissfeld JL, Kuller LH, Thaete F L, Evans RW, Hayes RB, Rosen CJ
Background & Aims: Insulin and insulin-like growth factor-I (IGF-I) affect proliferation, differentiation, and apoptosis and are potential risk factors for colorectal cancer (CRC). Visceral obesity, possibly via hyperinsulinemia, has also been linked to CRC risk. We evaluated the relationship of insulin, IGF-I, insulin-like growth factor binding protein (IGFBP) 3, and visceral adipose tissue (VAT) in subjects with adenomatous polyps, the precursor lesion of colorectal cancer. Methods: Participants were asymptomatic subjects who underwent screening flexible sigmoidoscopy (FSG) within the Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial. Subjects underwent single-slice, computerized tomography scanning to measure VAT and serum fasting insulin, IGF-I, and IGFBP-3 measurements. Results: Four hundred fifty-eight subjects were enrolled, of which 202 subjects had an adenoma, 70 of which were an advanced adenoma. IGF-I (P = .02), IGF-I/IGFBP-3 ratio (P = .003), and insulin (P = .02) were significantly increased in subjects with adenomas compared with controls. In an unadjusted logistic regression analysis using sex-specific quartile cut points, subjects in quartile 4 in comparison with quartile 1 of IGF-I (odds ratio [OR] = 1.7; [95% CI: 1.0–2.9], Ptrend = .03), IGF-I/IGFBP-3 ratio (OR = 1.9 [95% CI: 1.1–3.3], Ptrend = .01), and insulin (OR = 2.1 [95% CI: 1.2–3.6], Ptrend = .04) were at increased risk of adenoma. When limiting the case group to advanced adenomas, the effect was more pronounced: IGF-I (OR = 2.8 [95% CI: 1.3–6.2], Ptrend = .006), IGF-I/IGFBP-3 ratio (OR = 2.3, [95% CI: 1.0–5.2], Ptrend = .04), and insulin (OR = 2.3 [95% CI: 1.1–4.9], Ptrend = .14). Visceral adipose tissue was not associated with adenoma risk. Conclusions: Levels of IGF-I, ratio of IGF-I/IGFBP-3, and insulin are associated with adenomas and even more so with advanced adenomas. These data support the hypothesis that insulin and IGF-I may contribute to the development and advancement of adenomatous polyps.

Clinical—liver, pancreas, and biliary tract

Complementary Stimulation of Hepatobiliary Transport and Detoxification Systems by Rifampicin and Ursodeoxycholic Acid in Humans
Marschall H, Wagner M, Zollner G, Fickert P, Diczfalusy U, Gumhold J, Silbert D, Fuchsbichler A, Benthin L, Grundström R, Gustafsson U, Sahlin S, Einarsson C, Trauner M
Background & Aims: Rifampicin (RIFA) and ursodeoxycholic acid (UDCA) improve symptoms and biochemical markers of liver injury in cholestatic liver diseases by largely unknown mechanisms. We aimed to study the molecular mechanisms of action of these drugs in humans. Methods: Thirty otherwise healthy gallstone patients scheduled for cholestectomy were randomized to RIFA (600 mg/day for 1 week) or UDCA (1 g/day for 3 weeks) or no medication before surgery. Routine biochemistry, lipids, and surrogate markers for P450 activity (4?-hydroxy cholesterol, 4?-OH-C) and bile acid synthesis (7?-hydroxy-4-cholesten-3-one, C-4) were measured in serum. Bile acids were analyzed in serum, urine, and bile. A wedge liver biopsy specimen was taken to study expression of hepatobiliary ABC transporters as well as detoxification enzymes and regulatory transcription factors. Results: RIFA enhanced bile acid detoxification as well as bilirubin conjugation and excretion as reflected by enhanced expression of CYP3A4, UGT1A1, and MRP2. These molecular effects were paralleled by decreased bilirubin and deoxycholic acid concentrations in serum and decreased lithocholic and deoxycholic acid concentrations in bile. UDCA on the other hand stimulated the expression of BSEP, MDR3, and MRP4. UDCA became the predominant bile acid after UDCA treatment and lowered the biliary cholesterol saturation index. Conclusions: RIFA enhances bile acid detoxification as well as bilirubin conjugation and export systems, whereas UDCA stimulates the expression of transporters for canalicular and basolateral bile acid export as well as the canalicular phospholipid flippase. These independent but complementary effects may justify a combination of both agents for the treatment of cholestatic liver diseases.

Mortality and Hospital Utilization for Hepatocellular Carcinoma in the United States
Kim W R, Gores GJ, Benson JT, Therneau TM, Melton III L J
Background & Aims: The incidence of hepatocellular carcinoma (HCC) has been increasing in the United States. Although resource-intensive treatment modalities have been increasingly applied, these patients still have poor survival. We examined 2 nationally representative databases, the Multiple Cause of Death file and the Nationwide Inpatient Sample database, to examine trends in mortality and hospital service utilization related to HCC. Methods: In both databases, a priori criteria were used to identify cases of HCC. All other available diagnostic fields were examined to characterize coexistent liver disease. Age-, sex-, and race-specific mortality from HCC was calculated, and temporal changes in mortality rates were evaluated using the multivariable Poisson model. Hospital service utilization was estimated based on length of stay, total hospitalization charges, and principal procedures. Results: The age-, sex-, and race-specific mortality from HCC increased from 1.54 to 2.58 per 100,000 per year between 1980 and 1998. Male sex, African and Asian race, and increasing age were also associated with higher mortality. The estimated total charge for HCC hospitalizations nationwide increased from $241 million in 1988 to $509 million in 2000 after inflation adjustment. Commonly employed procedures in 2000 included angiography/embolization, resection, local ablative therapy, and liver transplantation. Conclusions: In the recent past, mortality and hospital service utilization related to HCC increased substantially. Closer epidemiologic surveillance to understand causation of HCC at the population level and to help implement primary and secondary prevention is urgently warranted.

Survival After Liver Transplantation in Patients With Hepatic Iron Overload: The National Hemochromatosis Transplant Registry
Kowdley KV, Brandhagen DJ, Gish RG, Bass NM, Weinstein J, Schilsky ML, Fontana RJ, McCashland T, Cotler SJ, Bacon BR, Keeffe EB, Gordon F, Polissar N
Background & Aims: Previous uncontrolled studies have suggested that patients with hepatic iron overload have a poor outcome after liver transplantation. We examined the effect of HFE mutations on posttransplantation survival in patients with hepatic iron overload. Methods: Two hundred sixty patients with end-stage liver disease and hepatic iron overload were enrolled from 12 liver transplantation centers. Hepatic iron concentration (HIC), hepatic iron index (HII), HFE mutation status, and survival after liver transplantation were recorded. Results:HFE-associated hemochromatosis (HH) defined as homozygosity for the C282Y (n = 14, 7.2%) mutation or compound heterozygosity for the C282Y/H63D (n = 11, 5.6%) mutation was identified in 12.8% of patients. Survival postliver transplantation was significantly lower among patients with HH (1-, 3-, and 5-year survival rates of 64%, 48%, 34%, respectively) compared with simple heterozygotes (C282Y/wt or H63D/wt) or wild-type patients. Patients with HH had a hazard ratio for death of 2.6 (P = .002) after adjustment for age, United Network for Organ Sharing status, year of transplantation, and either elevated HII or HIC. Non-HH patients with hepatic iron overload also had significantly decreased survival when compared with the overall population undergoing liver transplantation (OR = 1.4, 95% CI: 1.15–1.61, P < .001). Conclusions: One- and 5-year survivals after liver transplantation are significantly lower among patients with HFE-associated HH. Our data also suggest that hepatic iron overload may be associated with decreased survival after liver transplantation, even in patients without HH. Early diagnosis of hepatic iron overload using HFE gene testing and iron depletion prior to liver transplantation may improve posttransplantation survival, particularly among patients with HH.

Probability of Pancreatic Cancer Following Diabetes: A Population-Based Study
Chari ST, Leibson CL, Rabe KG, Ransom J, de Andrade M, Petersen GM
Background & Aims: Although diabetes occurs frequently in pancreatic cancer, the value of new-onset diabetes as a marker of underlying pancreatic cancer is unknown. Methods: We assembled a population-based cohort of 2122 Rochester, Minnesota, residents age ≥50 years who first met standardized criteria for diabetes between January 1, 1950, and December 31, 1994, and identified those who developed pancreatic cancer within 3 years of meeting criteria for diabetes. We compared observed rates of pancreatic cancer with expected rates based on the Iowa Surveillance Epidemiology and End Results registry. In a nested case control study, we compared body mass index (BMI) and smoking status in diabetes subjects with and without pancreatic cancer. Results: Of 2122 diabetic subjects, 18 (0.85%) were diagnosed with pancreatic cancer within 3 years of meeting criteria for diabetes; 10 of 18 (56%) were diagnosed <6 months after first meeting criteria for diabetes, and 3 were resected. The observed-to-expected ratio of pancreatic cancer in the cohort was 7.94 (95% CI, 4.70–12.55). Compared with subjects without pancreatic cancer, diabetic subjects with pancreatic cancer were more likely to have met diabetes criteria after age 69 (OR = 4.52, 95% CI, 1.61–12.74) years but did not differ significantly with respect to BMI values (29.2 ± 6.8 vs 26.5 ± 5.0, respectively). A larger proportion of those who developed pancreatic cancer were ever smokers (92% vs 69%, respectively), but this did not reach statistical significance. Conclusions: Approximately 1% of diabetes subjects aged ≥50 years will be diagnosed with pancreatic cancer within 3 years of first meeting criteria for diabetes. The usefulness of new-onset diabetes as marker of early pancreatic cancer needs further evaluation.

Drug-Induced Liver Injury: An Analysis of 461 Incidences Submitted to the Spanish Registry Over a 10-Year Period
Andrade RJ, Lucena M I, Fernández M C, Pelaez G, Pachkoria K, García-Ruiz E, García-Muñoz B, González-Grande R, Pizarro A, Durán JA, Jiménez M, Rodrigo L, Romero-Gomez M, Navarro JM, Planas R, Costa J, Borras A, Soler A, Salmerón J, Martin-Vivaldi R
Background & Aims: Progress in the understanding of susceptibility factors to drug-induced liver injury (DILI) and outcome predictability are hampered by the lack of systematic programs to detect bona fide cases. Methods: A cooperative network was created in 1994 in Spain to identify all suspicions of DILI following a prospective structured report form. The liver damage was characterized according to hepatocellular, cholestatic, and mixed laboratory criteria and to histologic criteria when available. Further evaluation of causality assessment was centrally performed. Results: Since April 1994 to August 2004, 461 out of 570 submitted cases, involving 505 drugs, were deemed to be related to DILI. The antiinfective group of drugs was the more frequently incriminated, amoxicillin-clavulanate accounting for the 12.8% of the whole series. The hepatocellular pattern of damage was the most common (58%), was inversely correlated with age (P < .0001), and had the worst outcome (Cox regression, P < .034). Indeed, the incidence of liver transplantation and death in this group was 11.7% if patients had jaundice at presentation, whereas the corresponding figure was 3.8% in nonjaundiced patients (P < .04). Factors associated with the development of fulminant hepatic failure were female sex (OR = 25; 95% CI: 4.1–151; P < .0001), hepatocellular damage (OR = 7.9; 95% CI: 1.6–37; P < .009), and higher baseline plasma bilirubin value (OR = 1.15; 95% CI: 1.09–1.22; P < .0001). Conclusions: Patients with drug-induced hepatocellular jaundice have 11.7% chance of progressing to death or transplantation. Amoxicillin-clavulanate stands out as the most common drug related to DILI.

Peginterferon-?-2a (40KD) and Ribavirin for 16 or 24 Weeks in Patients With Genotype 2 or 3 Chronic Hepatitis C
von Wagner M, Huber M, Berg T, Hinrichsen H, Rasenack J, Heintges T, Bergk A, Bernsmeier C, Häussinger D, Herrmann E, Zeuzem S
Background & Aims: Standard therapy of patients with chronic hepatitis C virus (HCV) infected with HCV genotype-2 or -3 is the combination of pegylated interferon-? and ribavirin for 24 weeks. Whether shorter treatment durations are possible for these patients without compromising sustained virologic response rates is unknown. Methods: Patients chronically infected with HCV-2 (n = 39), HCV-2/3 (n = 1), or HCV-3 (n = 113) were treated with peginterferon-?-2a (180 ?g/wk) plus ribavirin 800–1200 mg/day. HCV RNA was quantitatively assessed after 4 weeks. Patients with a rapid virologic response (HCV RNA below 600 IU/mL) were randomized for a total treatment duration of 16 (group A) or 24 weeks (group B). All patients with HCV RNA ≥600 IU/mL at week 4 (group C) were treated for 24 weeks. End-of-treatment and sustained virologic response were assessed by qualitative RT-PCR (sensitivity 50 IU/mL). Results: Only 11 of 153 patients (7%) were allocated to group C. End-of-treatment and sustained virologic response rates were 94% and 82%, (group A), 85% and 80% (group B), and 73% and 36% (group C), respectively. In patients infected with genotype HCV-3 and high viral load (>800,000 IU/mL), a significant lower sustained virologic response rate was found than in patients infected with HCV-3 and a viral load lower or equal to 800,000 IU/mL (59% vs 85%, respectively; P = .003). Conclusions: In HCV-2 and -3 (low viral load)-infected patients who have a rapid virologic response, treatment for 16 weeks with peginterferon-?-2a and ribavirin is sufficient. In patients infected by HCV-3 (high viral load), longer treatment may be necessary.

A 1-Year Trial of Telbivudine, Lamivudine, and the Combination in Patients With Hepatitis B e Antigen—Positive Chronic Hepatitis B
Lai CL, Leung N, Teo EK, Tong M, Wong F, Hann HW, Han S, Poynard T, Myers M, Chao G, Lloyd D, Brown NA
Background & Aims: A previous 4-week trial of telbivudine in patients with chronic hepatitis B indicated marked antiviral effects with good tolerability, leading to the present 1-year phase 2b trial. Methods: This randomized, double-blind, multicenter trial evaluated the efficacy and safety of telbivudine 400 or 600 mg/day and telbivudine 400 or 600 mg/day plus lamivudine 100 mg/day (Comb400 and Comb600) compared with lamivudine 100 mg/day in hepatitis B e antigen (HBeAg)-positive adults with compensated chronic hepatitis B. Results: A total of 104 patients were randomized 1:1:1:1:1 among the 5 groups. Median reductions in serum hepatitis B virus (HBV) DNA levels at week 52 (log10 copies/mL) were as follows: lamivudine, 4.66; telbivudine 400 mg, 6.43; telbivudine 600 mg, 6.09; Comb400, 6.40; and Comb600, 6.05. At week 52, telbivudine monotherapy showed a significantly greater mean reduction in HBV DNA levels (6.01 vs 4.57 log10 copies/mL; P < .05), clearance of polymerase chain reaction–detectable HBV DNA (61% vs 32%; P < .05), and normalization of alanine aminotransferase levels (86% vs 63%; P < .05) compared with lamivudine monotherapy, with proportionally greater HBeAg seroconversion (31% vs 22%) and less viral breakthrough (4.5% vs 15.8%) (P = NS for both). Combination treatment was not better than telbivudine alone. All treatments were well tolerated. In exploratory scientific analyses, clinical efficacy at 1 year appeared related to reduction in HBV DNA levels in the first 6 months of treatment. Conclusions: Patients with chronic hepatitis B treated with telbivudine exhibited significantly greater virologic and biochemical responses compared with lamivudine. Results with the combination regimens were similar to those obtained with telbivudine alone. These data support the ongoing phase 3 evaluation of telbivudine for treatment of patients with chronic hepatitis B.

Basic-alimentary tract

Functional Significance and Clinical Phenotype of Nontruncating Mismatch Repair Variants of MLH1
Raevaara TE, Korhonen MK, Lohi H, Hampel H, Lynch E, Lönnqvist KE, Holinski-Feder E, Sutter C, McKinnon W, Duraisamy S, Gerdes AM, Peltomäki P, Kohonen-Ccorish M, Mangold E, MacRae F, Greenblatt M, de la Chapelle A, Nyström M
Background & Aims: Germline mutations in mismatch repair genes are associated with hereditary nonpolyposis colorectal cancer. A significant proportion of mutations are nontruncating and associated with a variability of clinical phenotype and microsatellite instability and with occasional presence of residual protein in tumor tissue that suggests impaired functional activity but not total lack of mismatch repair. To address pathogenic significance and mechanism of pathogenicity, we studied the functionality of 31 nontruncating MLH1 mutations found in clinically characterized colorectal cancer families and 3 other variations listed in a mutation database. Methods: Mutations constructed by site-directed mutagenesis were studied for protein expression/stability, subcellular localization, protein-protein interaction, and repair efficiency. The genetic and biochemical data were correlated with clinical data. Finally, comparative sequence analysis was performed to assess the value of sequence homology as a tool for predicting functional results. Results: Altogether, 22 mutations were pathogenic in more than one assay, 2 variants were impaired in one assay, and 10 variants acted like wild-type protein. Twenty of 34 mutations affected the quantity of MLH1 protein, whereas only 15 mainly amino-terminal mutations were defective in an in vitro repair assay. Comparative sequence analysis correctly predicted functional studies for 82% of variants. Conclusions: Pathogenic nontruncating alterations in MLH1 may interfere with different biochemical mechanisms but generally more than one. The severe biochemical defects are mirrored by phenotypic characteristics such as early age at onset and high microsatellite instability, whereas variants with no or mild defects in functionality are associated with variable clinical phenotypes.

Interleukin-13 Is the Key Effector Th2 Cytokine in Ulcerative Colitis That Affects Epithelial Tight Junctions, Apoptosis, and Cell Restitution
Heller F, Florian P, Bojarski C, Richter J, Christ M, Hillenbrand B, Mankertz J, Gitter AH, Bürgel N, Fromm M, Zeitz M, Fuss I, Strober W, Schulzke JD
Background & Aims: Ulcerative colitis (UC) is characterized by a Th2 immune response with inflammation and epithelial barrier dysfunction. So far, Th2 cytokines have not been shown to directly influence epithelial barrier function. Methods: Lamina propria mononuclear cells (LPMCs) were stimulated and interleukin (IL)-13 was measured by enzyme-linked immunosorbent assay. Functional IL-13 and IL-4 effects were studied on HT-29/B6 colonic epithelial cells in Ussing chambers and by conductance scanning. Apoptosis was detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assays. IL-13/IL-4 receptors were analyzed by reverse-transcription polymerase chain reaction and immunofluorescence. Western blotting combined with immunofluorescence was used to detect tight junction proteins. Furthermore, restitution velocity was measured. Finally, mucosal biopsy specimens from patients with UC were compared with cultured cells for these features. Results: LPMCs from patients with UC produced large amounts of IL-13 (985 ± 73 pg/mL), much more than from controls or patients with Crohn’s disease. IL-13R?1 and IL-4R? receptors were present in HT-29/B6 cells and colonic epithelial cells of control patients and patients with UC. IL-13 had a dose-dependent effect on transepithelial resistance of HT-29/B6 monolayers (reduction to 60% ± 4%), whereas IL-4 had no effect. This was due to an increased number of apoptotic cells (5.6-fold ± 0.9-fold) and an increased expression of the pore-forming tight junction protein claudin-2 to 295% ± 37%, both of which contributed equally. Finally, epithelial restitution velocity decreased from 15.1 ± 0.6 to 10.6 ± 0.5 ?m/h after treatment with IL-13. Parallel changes were observed in human samples, with an increase in claudin-2 expression to 956% ± 252%. Conclusions: IL-13 was identified as an important effector cytokine in UC that impairs epithelial barrier function by affecting epithelial apoptosis, tight junctions, and restitution velocity.

Identification of Functional Genetic Variants in Cyclooxygenase-2 and Their Association With Risk of Esophageal Cancer
Zhang X, Miao X, Tan W, Ning B, Liu Z, Hong Y, Song W, Guo Y, Zhang X, Shen Y, Qiang B, Kadlubar FF, Lin D
Background & Aims: Overexpression of cyclooxygenase-2 (COX-2) is implicated in many steps of cancer development. Single nucleotide polymorphisms (SNPs) in the COX-2 promoter might contribute to differential COX-2 expression and subsequent interindividual variability in susceptibility to cancer. This study sought to identify functional SNPs in the COX-2 promoter and evaluated their effects on the risk of developing esophageal squamous cell carcinoma (ESCC). Methods: Thirty individual DNA samples were sequenced to search for SNPs, and the function of the SNPs was examined by a set of biochemical assays. Genotypes and haplotypes were analyzed in 1026 patients and 1270 controls, and odds ratios and 95% confidence intervals (CIs) were estimated by logistic regression. Results: Three SNPs, ?1290A?G, ?1195G?A, and ?765G?C, were identified; the frequencies of variant alleles were 0.04, 0.51, and 0.02, respectively. The ?1195G?A change creates a c-MYB binding site and displays a higher promoter activity. The ?1195A-containing haplotypes had significantly increased luciferase expression and COX-2 messenger RNA levels in esophageal tissues compared with the ?1195G-containing counterparts. A case-control analysis showed a 1.72-fold (95% CI, 1.35–2.20) and 2.24-fold (95% CI, 1.59–3.16) excess risk of developing ESCC for the ?1195AA or ?765CC genotype carriers compared with noncarriers. A greater risk of developing ESCC was observed for A?1195-C?765-containing haplotypes compared with G?1195-G?765-containing haplotypes, suggesting an interaction between the ?1195G?A and ?765G?C polymorphisms in the context of haplotype. Conclusions: These findings indicate that genetic variants in COX-2 may play a role in mediating susceptibility to esophageal cancer.

Oncogenic Potential of MEK1 in Rat Intestinal Epithelial Cells Is Mediated via Cyclooxygenase-2
Komatsu K, Buchanan F G, Katkuri S, Morrow JD, Inoue H, Otaka M, Watanabe S, DuBois RN
Background & Aims: The mitogen-activated protein kinase/extracellular signal-regulated protein kinase kinase (MEK) pathway plays an important role in the regulation of cell growth and differentiation. Constitutively active components of the MEK signaling cascade can induce oncogenic transformation in many cell systems. Downstream MEK signaling also plays an important role in the regulation of cyclooxygenase-2 (COX-2), which is known to be involved in colorectal cancer. Therefore, we determined the role of COX-2 on the oncogenic potential of MEK1 in nontransformed rat intestinal epithelial cells. Methods: Constitutively active MEK1 (CA-MEK) mutant transfected rat intestinal epithelial cells were established and tested for their ability to grow in soft agar and form tumors in vivo. The effect of CA-MEK on sodium butyrate (NaB)–induced apoptosis was evaluated by the Annexin V assay. The transcriptional activity and posttranscriptional stability of the COX-2 gene was determined by transient transfection with COX-2 reporter variants and by Northern analysis. To address the role of COX-2 in tumor growth in vivo, xenografted mice were treated with celecoxib (100 mg/kg) or vehicle. Results: CA-MEK transfected RIE-1 and IEC-6 cells formed colonies in soft agar and tumors in nude mice. These cells showed resistance to NaB-induced apoptosis and cell cycle arrest. MEK activation led to increased expression of COX-2, Bcl-XL, Mcl-1, and phosphorylated Bad and decreased expression of Bak. Along with elevated COX-2 levels, PGI2 and PGE2 levels were also increased. Pharmacologic inhibition of COX-2 inhibited MEK-induced tumor growth in vivo through enhanced apoptosis. Conclusions: COX-2 and its bioactive lipid products may play an important role in MEK-induced transformation.

Epidermal Growth Factor Partially Restores Colonic Ion Transport Responses in Mouse Models of Chronic Colitis
McCole DF, Rogler G, Varki N, Barrett KE
Background & Aims: Epidermal growth factor receptor (EGFR) activation, which plays an important role in regulating intestinal ion transport, can alleviate clinical symptoms such as diarrhea in patients with ulcerative colitis and promote mucosal restitution in animal models of colitis. Here, we investigate whether EGFR can regulate colonic ion transport in the setting of colitis. Methods: Distal colon from control mice and mice with colitis was retained for immunohistochemistry or mounted in Ussing chambers. Ion transport responses across the tissues to the calcium agonist carbachol and the adenosine 3?,5?-cyclic monophosphate agonist forskolin were measured with or without epidermal growth factor (EGF) pretreatment. Results: EGF pretreatment of normal colonic mucosa inhibited ion transport responses to carbachol and forskolin but potentiated the reduced ion transport responses seen in dextran sulfate sodium (DSS)-treated and mdr1a knockout mouse colon. Ion substitution studies and the sodium transport inhibitor amiloride showed that sodium movement primarily accounted for the potentiating effect of EGF in DSS-treated tissues, despite decreased sodium channel expression. EGF potentiation of transport responses in DSS-treated colon was completely blocked by the cytoskeletal disruptor cytochalasin D and the phosphatidylinositol 3-kinase inhibitor wortmannin, whereas the novel and conventional protein kinase C isoform inhibitor Gö6850 and the extracellular signal–regulated kinase inhibitor PD98059 partially reduced EGF effects. EGFR epithelial distribution and transforming growth factor ? expression were also altered in DSS-treated tissues. Conclusions: Chronic inflammation uncovers a potentiating effect of EGFR activation on epithelial electrogenic sodium absorption that would be expected to ameliorate diarrheal symptoms associated with colitis.

HB-EGF Enhances Restitution After Intestinal Ischemia/Reperfusion via PI3K/Akt and MEK/ERK1/2 Activation
El-Assal ON, Besner GE
Background & Aims: Early recovery of intestinal function after injury occurs by restitution, a complex process with a poorly understood molecular basis. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a potent chemotactic factor that is induced during ischemia/reperfusion in vivo and intestinal wounding in vitro. The role of HB-EGF in intestinal restitution and the underlying intracellular signaling pathways involved were investigated. Methods: Adult rats were subjected to intestinal ischemia, with histologic and biochemical damage assessed during the first 3 hours of reperfusion. The effect of recombinant HB-EGF (rHB-EGF) on structural and functional recovery of the intestine by restitution was evaluated in vivo. Scrape wounding of intestinal epithelial cell monolayers was used to elucidate the mechanisms of intrinsic and rHB-EGF-induced restitution. Results: Early structural recovery occurred within 3 hours of reperfusion and was attributed to restitution rather than proliferation. HB-EGF treatment significantly improved structural recovery and accelerated functional recovery of the gut barrier. In vivo restitution was preceded by activation of Akt and extracellular signal-regulated kinase (ERK) 1/2, which were accelerated and enhanced by HB-EGF treatment. Blocking of ErbB-1, phosphatidylinositol 3-kinase (PI3K)/Akt, or mitogen-activated protein kinase/ERK kinase (MEK)/ERK activity resulted in significant reduction in intrinsic and HB-EGF-induced restitution in vitro. Endogenous HB-EGF was shown to play an essential role in wound-induced ErbB-1 and ERK1/2 activation and in intrinsic restitution. Conclusions: Endogenous HB-EGF, ErbB-1, PI3K/Akt, and MEK/ERK are involved in intrinsic restitution. rHB-EGF enhances restitution in vivo and in vitro in a PI3K/Akt- and MEK/ERK1/2-dependent fashion.

Expression Pattern of Wnt Signaling Components in the Adult Intestine
Gregorieff A, Pinto D, Begthel H, Destrée O, Kielman M, Clevers H
Background & Aims: In the intestine, the canonical Wnt signaling cascade plays a crucial role in driving the proliferation of epithelial cells. Furthermore, aberrant activation of Wnt signaling is strongly associated with the development of colorectal cancer. Despite this evidence, little is known about the precise identity and localization of Wnts and their downstream effectors in the adult intestine. To address this issue, we examined the expression pattern of all Wnts, Frizzleds (Fzs), low-density lipoprotein receptor-related proteins, Wnt antagonists, and T-cell factors in the murine small intestine and colon and adenomas. Methods: Embryonic, postnatal, and adult intestinal samples were subjected to in situ hybridization by using specific RNA probes for the various genes tested. Results: Our analysis showed high expression of several signaling components (including Wnt-3, Wnt-6, Wnt-9b, Frizzled 4, Frizzled 6, Frizzled 7, low-density lipoprotein receptor-related protein 5, and secreted Frizzled-related protein 5) in crypt epithelial cells. We also detected Wnt-2b, Wnt-4, Wnt-5a, Wnt-5b, Frizzled 4, and Frizzled 6 in differentiated epithelial and mesenchymal cells of the small intestine and colon. Finally, several factors (Frizzled 4, T-cell factor 1, lymphoid enhancer factor, Dickkopf 2, Dickkopf 3, and Wnt-interacting factor) displayed differential expression in normal vs neoplastic tissue. Conclusions: Our study predicts a much broader role for Wnt signaling in gut development and homeostasis than was previously anticipated from available genetic studies and identifies novel factors likely involved in promoting canonical and noncanonical Wnt signals in the intestine.

Basic-liver, pancreas, and biliary tract

Tumor Necrosis Factor-? Mediates Pancreatitis Responses in Acinar Cells via Protein Kinase C and Proline-Rich Tyrosine Kinase 2
Satoh A, Gukovskaya AS, Edderkaoui M, Daghighian MS, Reeve Jr JR, Shimosegawa T, Pandol SJ
Background & Aims: Although tumor necrosis factor ? is implicated as an important mediator of the inflammatory response in acute pancreatitis, its role in other pathologic features of the disease remains unknown. We investigated the role for tumor necrosis factor ? in cytoskeletal responses and the underlying signaling mechanisms in pancreatic acinar cells. Methods: In isolated rat pancreatic acini and AR42J cells, we determined the effect of tumor necrosis factor ? on the actin cytoskeleton by rhodamine-phalloidin. Using pharmacological and molecular approaches, we assessed the involvement of protein kinase C, Src kinases, and proline-rich tyrosine kinase 2 in the process. We also studied the involvement of these signaling pathways in tumor necrosis factor-?-induced nuclear factor-?B activation and apoptosis. Results: Tumor necrosis factor-? increased the tyrosine phosphorylation of proline-rich tyrosine kinase 2 in acinar cells. The broad-spectrum protein kinase C inhibitor and the Src kinase inhibitor both inhibited tumor necrosis factor-?-induced proline-rich tyrosine kinase 2 phosphorylation, but at different tyrosine residues. Using protein kinase C isoform-specific inhibitors and the antisense approach, we showed that protein kinase C ? and ? mediate proline-rich tyrosine kinase 2 tyrosine phosphorylation. Tumor necrosis factor-? caused disorganization of the actin cytoskeleton by a mechanism dependent on protein kinase C, Src kinases, and proline-rich tyrosine kinase 2. Inhibition of protein kinase C, but not Src kinases, decreased tumor necrosis factor-?-induced apoptosis. Furthermore, with antisense transfections, we showed that protein kinase C ? and ?, but not proline-rich tyrosine kinase 2, mediate tumor necrosis factor ?-induced nuclear factor-?B activation. Conclusions: Tumor necrosis factor-? activates proline-rich tyrosine kinase 2 to cause cytoskeletal disorganization and nuclear factor-?B to cause inflammatory response, and it triggers cell death signaling through divergent mechanisms mediated by protein kinase C. The results provide insights into the mechanisms in pancreatic acinar cells that link tumor necrosis factor ? to critical processes in acute pancreatitis.

Free Radical-Dependent Dysfunction of Small-for-Size Rat Liver Grafts: Prevention by Plant Polyphenols
Zhong Z, Connor HD, Froh M, Bunzendahl H, Lind H, Lehnert M, Mason RP, Thurman RG, Lemasters JJ
Background&Aims: The mechanisms by which small-for-size liver grafts decrease survival remain unclear. This study investigated the role of free radicals in injury to small-for-size grafts. Methods: Rat liver explants were reduced in size ex vivo and transplanted into recipients of the same or greater body weight, resulting in a graft weight and standard liver weight of approximately 50% and 25%, respectively. A polyphenol extract from Camellia sinenesis (20 ?g/mL) or an equivalent concentration of epicatechin was added to the storage solution and the lactated Ringer poststorage rinse solution. Results: Serum alanine aminotransferase release increased from approximately 60 U/L before implantation to 750, 1410, and 2520 U/L after full-size, half-size, and quarter-size transplantation, respectively. Total bilirubin increased slightly after transplantation of full-size and half-size grafts but increased 104-fold in quarter-size grafts. In quarter-size grafts, histological changes included necrosis, leukocyte infiltration, and eosinophilic inclusion body formation. Polyphenol treatment ameliorated these effects by ≥67%. Survival was 30% after transplantation of small-for-size grafts. After polyphenol treatment, survival increased to 70%. Free radicals in bile assessed by spin trapping and 4-hydroxynonenal adducts measured by immunohistochemistry were also greater in reduced-size grafts, an effect ameliorated by polyphenols. Epicatechin, a major polyphenol from Camellia sinenesis, also improved graft function and decreased enzyme release, histopathologic changes, and free radical formation. Conclusions: Increased formation of free radicals occurs after transplantation of reduced-size livers, which contributes to graft dysfunction and failure. Plant polyphenols decrease liver graft injury and increase survival of small-for-size liver grafts, most likely by scavenging free radicals.

A Proinflammatory, Antiapoptotic Phenotype Underlies the Susceptibility to Acute Pancreatitis in Cystic Fibrosis Transmembrane Regulator (?/?) Mice
Dimagno MJ, Lee SH, Hao Y, Zhou SY, McKenna BJ, Owyang C
Background & Aims: Cystic fibrosis transmembrane regulator (CFTR) gene mutations are associated with pancreatic insufficiency and pancreatitis. Chronic pancreatitis, including cystic fibrosis-related disease, may exist as a continuum between acute and chronic disease and may manifest as recurrent pain. We hypothesized that cftrm1UNC (?/?) mice, which have no evidence of chronic pancreatitis, are susceptible to developing acute pancreatitis.Methods: We used a cerulein hyperstimulation model of acute pancreatitis and measured histological changes, tissue edema, neutrophil infiltration, inflammatory mediators’ mRNA expression, apoptosis markers, and pancreatic trypsin and serum lipase activities. Additionally, we quantitated in vivo pancreatic secretion and pancreatic digestive enzymes. Results: Multiple proinflammatory cytokine genes were constitutively overexpressed in cftr (?/?) pancreas compared with wild-type mice. During acute pancreatitis, cftr (?/?) mice developed more severe acute pancreatitis than wild-type, as indicated by greater pancreatic edema, neutrophil infiltration, mRNA expression of multiple inflammatory mediators, and less apoptotic cell death. In contrast to wild-type mice, cftr (?/?) mice had blunted increases in pancreatic trypsin and serum lipase activities, but similar percentages of pancreatic trypsinogen activation. Finally, cftr (?/?) mice had less in vivo pancreatic secretion in response to cholecystokinin octapeptide and reduced pancreatic digestive enzyme protein and mRNA levels, thus suggesting mild pancreatic insufficiency. Conclusions: A baseline proinflammatory state and an antiapoptotic phenotype may sensitize cftr (?/?) mice to developing more severe acute pancreatitis with an exuberant pancreatic inflammatory response. Cftr (?/?) mice have mild pancreatic insufficiency, which partially explains the blunted increase of pancreatic and serum digestive enzymes during acute pancreatitis. These findings may explain the susceptibility to acute pancreatitis in persons with classic and nonclassic cystic fibrosis.

Cholangiocyte Endothelin 1 and Transforming Growth Factor ?1 Production in Rat Experimental Hepatopulmonary Syndrome
Luo B, Tang L, Wang Z, Zhang J, Ling Y, Feng W, Sun JZ, Stockard CR, Frost AR, Chen YF, Grizzle WE, Fallon MB
Background & Aims: Hepatic production and release of endothelin 1 plays a central role in experimental hepatopulmonary syndrome after common bile duct ligation by stimulating pulmonary endothelial nitric oxide production. In thioacetamide-induced nonbiliary cirrhosis, hepatic endothelin 1 production and release do not occur, and hepatopulmonary syndrome does not develop. However, the source and regulation of hepatic endothelin 1 after common bile duct ligation are not fully characterized. We evaluated the sources of hepatic endothelin 1 production after common bile duct ligation in relation to thioacetamide cirrhosis and assessed whether transforming growth factor ?1 regulates endothelin 1 production. Methods: Hepatopulmonary syndrome and hepatic and plasma endothelin 1 levels were evaluated after common bile duct ligation or thioacetamide administration. Cellular sources of endothelin 1 were assessed by immunohistochemistry and laser capture microdissection of cholangiocytes. Transforming growth factor ?1 expression and signaling were assessed by using immunohistochemistry and Western blotting and by evaluating normal rat cholangiocytes. Results: Hepatic and plasma endothelin 1 levels increased and hepatopulmonary syndrome developed only after common bile duct ligation. Hepatic endothelin 1 and transforming growth factor ?1 levels increased over a similar time frame, and cholangiocytes were a major source of each peptide. Transforming growth factor ?1 signaling in cholangiocytes in vivo was evident by increased phosphorylation and nuclear localization of Smad2, and hepatic endothelin 1 levels correlated directly with liver transforming growth factor ?1 and phosphorylated Smad2 levels. Transforming growth factor ?1 also stimulated endothelin 1 promoter activity, expression, and production in normal rat cholangiocytes. Conclusions: Cholangiocytes are a major source of hepatic endothelin 1 production during the development of hepatopulmonary syndrome after common bile duct ligation, but not in thioacetamide-induced cirrhosis. Transforming growth factor ?1 stimulates cholangiocyte endothelin 1 expression and production. Cholangiocyte-derived endothelin 1 may be an important endocrine mediator of experimental hepatopulmonary syndrome.

Autophagic Cell Death of Pancreatic Acinar Cells in Serine Protease Inhibitor Kazal Type 3—Deficient Mice
Ohmuraya M, Hirota M, Araki M, Mizushima N, Matsui M, Mizumoto T, Haruna K, Kume S, Takeya M, Ogawa M, Araki K, Yamamura K
Background & Aims: Serine protease inhibitor Kazal type 1 (SPINK1), which is structurally similar to epidermal growth factor, is thought to inhibit trypsin activity and to prevent pancreatitis. Point mutations in the SPINK1 gene seem to predispose humans to pancreatitis; however, the clinical significance of SPINK1 mutations remains controversial. This study aimed to elucidate the role of SPINK1. Methods: We generated Spink3-deficient (Spink3?/?) mice by gene targeting in mouse embryonic stem cells. Embryonic and neonatal pancreases were analyzed morphologically and molecularly. Specific probes were used to show the typical autophagy that occurs during acinar cell death. Results: In Spink3?/? mice, the pancreas developed normally up to 15.5 days after coitus. However, autophagic degeneration of acinar cells, but not ductal or islet cells, started from day 16.5 after coitus. Rapid onset of cell death occurred in the pancreas and duodenum within a few days after birth and resulted in death by 14.5 days after birth. There was limited inflammatory cell infiltration and no sign of apoptosis. At 7.5 days after birth, residual ductlike cells in the tubular complexes strongly expressed pancreatic duodenal homeodomain-containing protein 1, a marker of pancreatic stem cells, without any sign of acinar cell regeneration. Conclusions: The progressive disappearance of acinar cells in Spink3?/? mice was due to autophagic cell death and impaired regeneration. Thus, Spink3 has essential roles in the maintenance of integrity and regeneration of acinar cells.

Case report

Life-Threatening Chronic Enteritis Due to Colonization of the Small Bowel With Stenotrophomonas maltophilia
Hellmig S, Ott S, Musfeldt M, Kosmahl M, Rosenstiel P, Stüber E, Hampe J, Fölsch UR, Schreiber S
Chronic diarrheal illness and malabsorption are challenging diagnostic and clinical problems. The identification of the causative pathogens that are involved in gastrointestinal infections is often difficult. It took 85 years after the first description of a case of intestinal lipodystrophy by Georg Whipple in 1907 until the causative bacterium was characterized by using molecular genetics techniques. We here report the complicated clinical course of a young patient with chronic diarrhea accompanied by severe, life-threatening malabsorption with extensive weight loss. Histology and glucose hydrogen breath test were suggestive of a bacterial overgrowth syndrome in the small bowel, but standard culture-based techniques and serology failed to identify the causative bacteria. Thus, bacterial ribosomal DNA (16S ribosomal DNA) was extracted from duodenal biopsy samples and analyzed by community fingerprinting and species-specific polymerase chain reaction. Stenotrophomonas maltophilia was identified as the cause of chronic infectious enteritis. Only specific long-term antibiotic treatment with co-trimoxazole had a durable clinical effect and led to normalization of 16S ribosomal DNA profiles. This case shows the role of rare and uncommon bacteria in refractory and chronic human gastrointestinal infections. Genomic techniques, including 16S-based single-strand conformation polymorphism analysis, will play an increasing role in the diagnosis of chronic infections with facultatively pathogenic bacteria or in the clinical analysis of complex bacterial communities such as the intestinal bacterial microflora. Future enhancements in detection techniques will show that chronic bacterial infections are more frequent as a cause of gastrointestinal malfunction than commonly thought.

Microarrays and other new technologies

Analysis of the Biliary Transcriptome in Experimental Biliary Atresia
Carvalho E, Liu C, Shivakumar P, Sabla G, Aronow B, Bezerra JA
Background & Aims: Discovery of the pathogenic mechanisms of biliary atresia has been limited by the inability to study extrahepatic biliary tissues from patients at early phases of disease. Here, we used a rotavirus-induced model of biliary atresia to investigate the entire biliary transcriptome for molecular networks activated at the onset and different phases of progression to duct obstruction. Methods: We injected Balb/c mice with saline or rotavirus intraperitoneally within 24 hours of birth, microdissected the gallbladder and extrahepatic bile ducts en bloc 3, 7, and 14 days later, generated biotinylated RNA pools, and hybridized them against microarrays containing 45,101 gene products. Results: Data filtering, cluster analysis, and functional assignment of the gene expression platform revealed 2 unique patterns of expression. The first was an overarching expression of genes regulating immunity, enzymes, and structural proteins at all phases of atresia. Within this pattern, the sequential expression of the interferon inducers Irf7 and Irf9 at the onset of injury, and interferon-? and interferon-?–activated genes (Stat1, Igtp, Cxcl9, Cxcl10) at the time of duct obstruction, pointed to a prominent proinflammatory circuit. The second was the time-restricted expression of genes regulating biological networks previously unrecognized in biliary atresia, such as the complement components C3ar-1 and C1q-?/?. Conclusions: The coordinate expression of functionally related genes in the biliary transcriptome underscores a predominant proinflammatory footprint and provides a basis for identification of gene groups that may play regulatory roles in the pathogenesis of duct injury and obstruction in experimental biliary atresia.

Special report and review

New Insights Into the Genetic Regulation of Intestinal Cholesterol Absorption
Lammert F, Wang DQ H
pages 718-734The small intestine is a unique organ providing dietary and reabsorbed biliary cholesterol to the body. However, the molecular mechanisms whereby cholesterol is absorbed have not yet been fully understood. Recent research suggests that the newly identified Niemann-Pick C1-like 1 protein (NPC1L1) is expressed at the apical surface of enterocytes and plays a critical role in the absorption of intestinal cholesterol. Furthermore, adenosine triphosphate (ATP)-binding cassette (ABC) transporters ABCG5 and ABCG8 represent apical sterol export pumps that promote active efflux of cholesterol and plant sterols from enterocytes back into the intestinal lumen for excretion. This provides an explanation why cholesterol absorption is a selective process, with plant sterols and other noncholesterol sterols being absorbed poorly or not at all. These findings strongly support the concept that cholesterol absorption is a multistep process, which is regulated by multiple genes at the enterocyte level. The absorption efficiency of cholesterol is most likely determined by the net effect between influx and efflux of intraluminal cholesterol molecules across the brush border of the enterocyte. Combination therapy using a novel, specific, and potent cholesterol absorption (NPC1L1) inhibitor (ezetimibe) and HMG-CoA reductase inhibitors (statins) offers an efficacious new approach to the prevention and treatment of hypercholesterolemia.

Copyright © 2001-2005 by the American Gastroenterological Association. All rights reserved.

---

JOURNAL OF HEPATOLOGY

Table of Contents for August 2005 (Vol. 43, Issue 2)

Viral Hepatitis

Viral kinetics in patients with lamivudine-resistant hepatitis B during adefovir–lamivudine combination therapy, 16 May 2005
Mihm U, Gärtner BC, Faust D, Hofmann WP, Sarrazin C, Zeuzem S, Herrmann E
pages 217-224
Background/Aims
Mathematical analysis of viral kinetics during lamivudine-adefovir combination therapy has not yet been performed in patients with lamivudine-resistant hepatitis B.
Methods
In 8 patients with lamivudine-resistant hepatitis B, adefovir dipivoxil (10mg/day) was added to ongoing lamivudine. Viral decay during the first 8 weeks of combination therapy was described by a biphasic model to determine the efficacy: ?, of blocking viral production, the clearance: c, of free virus, and the loss of infected cells: ?.
Results
Median ? was 98%, median c was 0.7/day, and median ? was 0.07/day. No significant association was found between viral kinetic and baseline parameters and virologic and biochemical treatment response. When compared with viral kinetic constants reported for higher dose adefovir dipivoxil monotherapy, ? was lower (P=0.026) and ? was higher (P=0.008) in this study whereas c did not differ between both studies.
Conclusions
Although a recent study did not show any differences in the reduction of HBV DNA comparing monotherapy with adefovir dipivoxil to adefovir–lamivudine combination therapy in patients with lamivudine-resistant chronic hepatitis B, mathematical analysis of early viral kinetics suggests an additional effect of lamivudine on the infected cell loss during adefovir–lamivudine combination therapy.

Inhibition of hepatitis C virus translation and subgenomic replication by siRNAs directed against highly conserved HCV sequence and cellular HCV cofactors, 16 May 2005
Korf M, Jarczak D, Beger C, Manns MP, Krüger M
pages 225-234
Background/Aims
Small interfering RNAs (siRNAs) are an efficient tool to specifically inhibit gene expression by RNA interference. Since hepatitis C virus (HCV) replicates in the cytoplasm of liver cells without integration into the host genome, RNA-directed antiviral strategies are likely to successfully block the HCV replication cycle. Additional benefit might arise from inhibition of cellular cofactors of HCV replication, such as proteasome ?-subunit 7 (PSMA7) or Hu antigen R (HuR).
Methods
In this study, we investigated direct and cofactor-mediated inhibition of HCV by a panel of DNA-based retroviral vectors expressing siRNAs against highly conserved HCV sequences or the putative HCV cofactors PSMA7 and HuR. Effects were determined in HCV IRES-mediated translation assays and subgenomic HCV replicon cells.
Results
PSMA7- and HuR-directed siRNAs successfully inhibited expression of the endogenous genes, and PSMA7 and HuR silencing significantly diminished HCV replicon RNA and NS5B protein levels. HCV-directed siRNAs substantially inhibited HCV IRES-mediated translation and subgenomic HCV replication. Combinations of PSMA7- and HuR-directed siRNAs with HCV-directed siRNAs revealed additive HCV RNA inhibitory effects in monocistronic replicon cells.
Conclusions
A dual approach of direct- and cofactor-mediated inhibition of HCV replication might avoid selection of mutants and thereby become a powerful strategy against HCV.

Serum adiponectin correlates with viral characteristics but not histologic features in patients with chronic hepatitis C, 16 May 2005
Liu CJ, Chen PJ, Jeng YM, Huang WL, Yang WS, Lai MY, Kao JH, Chen DS
pages 235-242
Background/Aims
Adiponectin induces insulin sensitivity and modulates inflammatory responses. We thus studied the implications of adiponectin in patients with chronic hepatitis C virus (HCV) infection inherently linked to insulin resistance.
Methods
We analyzed the association of serum adiponectin levels with clinical, virologic, and histologic findings in 95 na?ve Taiwanese patients with chronic hepatitis C before and after antiviral therapy.
Results
At baseline, 14 (15%) of the 95 patients were obese and 26 (27%) had type 2 diabetes mellitus. Fifty-seven patients were infected with HCV genotype 1 and 38 with genotype 2. Steatosis and periportal fibrosis was present in 44 (46%) and 69 (73%), respectively. In multivariate analysis, male gender, insulin resistance, high HCV load and genotype 2 were significantly associated with a lower serum adiponectin level. In contrast, intrahepatic gene expression of adiponectin receptors was higher in genotype 2 compared with genotype 1. Serum adiponectin level did not correlate with other clinical or histologic parameters. After treatment, change of steatosis also did not correlate with the change of adiponectin level (P=0.61).
Conclusions
Adiponectin correlated with hepatitis C viral factors at both serum and liver tissue levels. The interactions among adiponectin, insulin resistance and chronic HCV infection merit further studies.

Peginterferon alfa-2b and ribavirin for treatment-refractory chronic hepatitis C, 25 May 2005
Krawitt EL, Ashikaga T, Gordon SR, Ferrentino N, Ray MA, Lidofsky SD, for the New York New England Study Team 
pages 243-249
Background/Aims: Treatment regimens with pegylated interferons have yielded improved response rates, compared with conventional interferon-based regimens, for chronic hepatitis C. However, little is known about the utility of such regimens for individuals who failed to respond to prior conventional interferon-based treatment.Methods: 182 patients who had previously failed to eliminate circulating hepatitis C virus 24 weeks after completion of a multi-week course of either interferon monotherapy or interferon in combination with ribavirin were treated with peginterferon alfa-2b weekly and ribavirin daily for 48 weeks.Results: The sustained viral response, was 20% (23/116) in previous non-responders and 55% (36/66) in previous relapsers (P<0.001). In previous non-responders, the sustained viral response in those with viral genotype 1 was 17% (19/109) compared to 57% (4/7) in those with genotypes 2 and 3 (P=0.03). In previous relapsers, the sustained viral response in those with viral genotype 1 was 53% (26/49) compared to 59% (10/17) with genotypes 2 and 3 (P=0.78).Conclusions: The response to pegylated interferon and ribavirin in previous non-responders with genotypes 2 and 3 and in prior relapsers with chronic hepatitis C is comparable to overall sustained viral response rates seen in previously untreated patients.

International, multicenter, randomized, controlled study comparing dynamically individualized versus standard treatment in patients with chronic hepatitis C, 3 June 2005
Zeuzem S, Pawlotsky JM, Lukasiewicz E, von Wagner M, Goulis I, Lurie Y, Gianfranco E, Vrolijk JM, Esteban JI, Hezode C, Lagging M, Negro F, Soulier A, Verheij-Hart E, Hansen B, Tal R, Ferrari C, Schalm SW, Neumann AU, for the DITTO-HCV Study Group 
pages 250-257
Background/Aims
The aim of this study was to increase virologic response rates by individualized treatment according to the early virologic response.
Methods
Serum HCV-RNA was frequently quantified in patients with chronic hepatitis C (n=270) treated with peginterferon alfa-2a (180?g/week) and ribavirin (1000–1200mg/day). After 6 weeks patients were classified as rapid (RVR), slow (SPR), flat (FPR), or null responders (NUR) and randomized within each viral kinetic class to continue therapy either with an individualized or standard regimen. Individualized therapy comprised peginterferon monotherapy (48 weeks) or shorter combination therapy (24 weeks) for RVR, triple therapy with histamine (1mg/day) (48 weeks) or prolonged combination therapy (72 weeks) for SPR, triple therapy for FPR, and high-dose peginterferon (360?g/week) plus ribavirin for NUR patients.
Results
Patients were categorized as RVR (n=171), SPR (n=65), FPR (n=10), or NUR (n=22). Overall end-of-treatment and sustained virologic response rates were 77 and 60% in the individualized and 77 and 66% in the standard treatment arm, respectively. Histamine in addition to peginterferon and ribavirin and high-dose peginterferon plus ribavirin did not improve virologic response rates in patients with FPR and NUR, respectively.
Conclusions
An improvement in virologic efficacy was not achieved with the available individualized treatment options.

Cirrhosis and its Complications

Up-regulation of nNOS and associated increase in nitrergic vasodilation in superior mesenteric arteries in pre-hepatic portal hypertension, 16 May 2005
Jurzik L, Froh M, Straub RH, Schölmerich J, Wiest R
pages 258-265
Background/Aims
Splanchnic arterial vasodilation in portal hypertension has been attributed largely to vascular NO overproduction. Three NO-synthase (NOS) isoforms have been identified of which e(ndothelial)-NOS has been found up-regulated and i(nducible)-NOS not expressed in the splanchnic circulation in portal hypertension. So far, n(euronal)-NOS has not been investigated and hence, the current study evaluates nNOS-expression and nNOS-mediated vasorelaxation in a model of portal vein-ligated rats (PVL).
Methods
Mesenteric vasculature of PVL and sham rats was evaluated for nNOS-protein (immunohistochemically and Western blotting). In vitro perfused de-endothelialized mesenteric arterial vasculature was pre-constricted with norepinephrine (EC80) and tested for nNOS-mediated vasorelaxation by periarterial nerve stimulation (PNS, 2–12Hz, 45V) before and after incubation with the NOS-inhibitor l-NAME (10?4M).
Results
nNOS was localized to the adventitia of the mesenteric arterial tree showing more intense staining and increased protein expression in PVL as compared to sham rats. PNS induced a frequency-dependent vasorelaxation, which was more pronounced in PVL rats. l-NAME abolished this difference in nerval-mediated vasorelaxation, the effect being significantly greater in PVL than in sham animals.
Conclusions
Perivascular nNOS-protein expression is enhanced in mesenteric arteries in portal hypertension mediating an increased nerval NO-mediated vasorelaxation. This nNOS-derived NO overproduction may play an important role in the pathogenesis of arterial vasodilation in portal hypertension.

Maintained cerebral and skeletal muscle oxygenation during maximal exercise in patients with liver cirrhosis, 16 May 2005
Bay Nielsen H, Secher NH, Clemmesen O, Ott P
pages 266-271
Background/Aims
In cirrhotic patients, insufficient redistribution of blood from splanchnic organs to the central circulation could limit blood supply to skeletal muscles and the brain during exercise.
Methods
Eight cirrhotic patients performed incremental cycling to exhaustion (74 (49–123) W; median with range).
Results
Heart rate increased from 68 (62–88)beats/min at rest to 142 (116–163)beats/min, cardiac output from 5.1 (3.3–7.2) to 12.9 (8.5–15.9)l/min, and mean arterial pressure from 89 (75–104) to 115 (92–129)mmHg (P<0.05), while the indocyanine green elimination determined hepatosplanchnic blood flow declined from 0.97 (0.55–1.46) to 0.62 (0.36–1.06)l/min (P<0.05). As assessed by near-infrared spectrophotometry, cerebral oxygenation (NIRS) was 61% (48–85%) and increased to 72% (57–86%) during exercise (P<0.05). The NIRS determined oxygenation of the vastus lateralis muscle also increased: the concentrations of oxygenated haemoglobin by 5.9 (0.57–9.47)?mol/l, deoxygenated haemoglobin by 7.2 (1.8–12.0)?mol/l, and thus total haemoglobin by 12.1 (3.6–21.5)?mol/l (P<0.05).
Conclusions
In patients with cirrhosis, exercise reduces hepatosplanchnic blood flow, while O2 supply to muscle and brain appears to increase indicating that blood redistribution from splanchnic organs does not limit blood flow to working muscles and the brain.

Genomic and functional characterization of stellate cells isolated from human cirrhotic livers, 9 May 2005
Sancho-Bru P, Bataller R, Gasull X, Colmenero J, Khurdayan V, Gual A, Nicolás JM, Arroyo V, Ginès P
pages 272-282
Background/Aims
Hepatic stellate cells (HSCs) are believed to participate in liver fibrogenesis and portal hypertension. Knowledge on human HSCs is based on studies using HSCs isolated from normal livers. We investigated the phenotypic, genomic and functional characteristics of HSCs from human cirrhotic livers.
Methods
HSC were obtained from normal and cirrhotic human livers. Cells were characterized by immunocytochemistry and gene microarray analysis. Cell proliferation, Ca2+ changes and cell contraction were assessed by 3H-thymidine incorporation and by using an epifluorescence microscope.
Results
HSCs freshly isolated from human cirrhotic livers showed phenotypical features of myofibroblasts. These features were absent in HSCs freshly isolated from normal human livers and become prominent after prolonged culture. HSCs from cirrhotic human livers markedly express genes involved in fibrogensis, inflammation and apoptosis. HSCs from normal livers after prolonged culture preferntially expressed genes related to fibrogenesis and contractility. Agonists induced proliferation, Ca2+ increase and cell contraction in HSCs isolated from human cirrhotic livers. Response to agonists was more marked in culture-activated HSCs and was not observed in HSCs freshly isolated from normal livers.
Conclusions
HSCs from human cirrhotic livers show fibrogenic and contractile features. However, the current model of HSCs activated in culture does not exactly reproduce the activated phenotype found in cirrhotic human livers.

Transplantation

Failure of hepatitis B vaccination in patients receiving lamivudine prophylaxis after liver transplantation for chronic hepatitis B, 18 May 2005
Lo CM, Liu CL, Chan SC, Lau GK, Fan ST
pages 283-287
Background/Aims
Lamivudine prophylaxis against hepatitis B virus (HBV) reinfection after liver transplantation is associated with recurrence due to escape mutants.
Methods
Fifty-two patients on lamivudine prophylaxis at a median of 412 days (median, 370–2040 days) after transplantation for chronic HBV-related liver disease received two courses of an accelerated schedule of double-dose recombinant HBV vaccine. Before vaccination, all patients were seronegative for HBsAg, anti-HBs and HBV DNA (by qPCR). Three intramuscular doses of vaccine (40?g each) were administered monthly and another identical course was repeated after 3 months. Lamivudine (100mg/day) was continued throughout the study.
Results
After the first course, two patients developed a weak response (anti-HBs titre of 12mIU/mL) that disappeared rapidly. One early responder developed anti-HBs (27mIU/mL) again after the second course but the other did not. Two other patients developed response (anti-HBs titre of 17 and 103mIU/mL, respectively) giving an overall response rate of 7.7%. The antibody level declined rapidly. At the end of the study, one patient who did not respond had developed viral breakthrough which was treated with adefovir dipivoxil therapy.
Conclusions
Active immunization with two courses of double-dose recombinant HBV vaccine has limited efficacy in patients receiving lamividine prophylaxis after liver transplantation.

Liver Failure, Growth and Cancer

Overexpression of the two nucleotide excision repair genes ERCC1 and XPC in human hepatocellular carcinoma, 26 April 2005
Fautrel A, Andrieux L, Musso O, Boudjema K, Guillouzo A, Langouët S
pages 288-293
Background/Aims
Little is known about the nucleotide excision repair (NER) pathway in the resistance of human hepatocellular carcinoma (HCC) to chemotherapeutics. We investigated expression of several NER genes in human HCC and matching non-tumor tissue (NT) and in normal liver.
Methods
Expression of CSA, CSB, XPC, hHR23B, XPA, XPB, ERCC1 and p53 genes was analyzed by quantitative RT-PCR and immunoblotting in 26 HCC and 9 normal livers.
Results
The seven NER genes and p53 were frequently overexpressed in HCC compared to matched NT. XPA, XPC, hHR23B and ERCC1 mRNA levels were significantly increased (p<0.05) in HCC arising in cirrhotic livers compared to non fibrotic tissue. Moreover, expression of ERCC1, XPA and XPC mRNA was significantly augmented in HCC, even more in tumors arising in cirrhotic liver. ERCC1, XPC ad XPA mRNA levels were highly correlated in NT and HCC. XPC and ERCC1 protein levels were also increased in HCC.
Conclusions
Our findings strongly suggest that overexpression of two key genes involved in the early steps of the NER process, ERCC1 and XPC, is associated with liver fibrogenesis and cancer and could be related to the well recognized resistance of HCC to chemotherapeutics.

C/EBP? contributes to hepatocyte growth factor-induced replication of rodent hepatocytes, 3 May 2005
Wang B, Gao C, Ponder KP
pages 294-302
Hepatocyte replication can be induced in vivo by hepatocyte growth factor (HGF), which might be used for gene therapy or to promote liver regeneration. However, the biochemical steps critical for this process are not clear. C/EBP? and C/EBP? are liver-enriched transcription factors that induce and inhibit hepatocyte replication, respectively. Because of their role in hepatocyte replication, this study examined the effect of HGF upon C/EBP proteins in vivo.
Methods
Rats were treated with HGF, and the effect upon C/EBPs was evaluated in liver extracts. Normal or C/EBP?-deficient mice were treated with HGF, and the effect upon hepatocyte replication was determined.
Results
HGF had no effect in rat liver upon C/EBP? or C/EBP? mRNA, nuclear protein, or nuclear DNA binding activity. However, HGF increased phosphorylated p90-RSK and ERK to18- and 3-fold normal, respectively. These kinases phosphorylate C/EBP? and increase its transcriptional activity. The percentage of hepatocytes that replicated in C/EBP?-deficient mice after HGF administration was only 1.1%, which was lower than the value of 6.6% for hepatocytes from HGF-treated normal mice (P=0.005).
Conclusions
C/EBP? contributes to the induction of hepatocyte replication in response to HGF in rodents, which is likely due to post-translational modifications.

A multicenter assessment of liver toxicity by MRI and biopsy in IBD patients on 6-thioguanine, 26 May 2005
Seiderer J, Zech CJ, Reinisch W, Lukas M, Diebold J, Wrba F, Teml A, Chalupna P, Stritesky J, Schoenberg SO, Schima W, Göke B, Ochsenkühn T
pages 303-309
Background/Aims
Although 6-thioguanine (6-TG) has been suggested as an effective treatment option for patients with inflammatory bowel disease (IBD), the recent description of its hepatotoxicity has led to the recommendation not to consider this drug. We initiated a multicenter safety study in IBD-patients treated with 6-TG to investigate hepatic changes by liver biopsy and magnetic resonance imaging (MRI).
Methods
Forty-five patients from three European centers treated with 6-TG (40–80mg/d) at least for 8 weeks were enrolled. In all patients liver biopsy and MRI were performed. Slides and MR images were independently read by two pathologists and radiologists, respectively, and interpreted according to predefined criteria by consent.
Results
In 8 patients nodular regenerative hyperplasia (NRH) was diagnosed by liver biopsy, in 8 additional patients NRH could not be excluded due to equivocal pathological findings. MRI demonstrated a sensitivity of 77% and a specificity of 72% in the detection of pathohistological findings consistent with and/or possibly related to NRH.
Conclusions
Our study suggests that 6-TG therapy in IBD patients is associated with NRH of the liver. Based on a special MRI protocol, non-invasive diagnosis of NRH with promising sensitivity and specificity was demonstrated.

Tumor doubling time predicts recurrence after surgery and describes the histological pattern of hepatocellular carcinoma on cirrhosis, 16 May 2005
Cucchetti A, Vivarelli M, Piscaglia F, Nardo B, Montalti R, Grazi GL, Ravaioli M, Barba GL, Cavallari A, Bolondi L, Pinna AD
pages 310-316
Background/Aims
Recurrence of hepatocellular carcinoma (HCC) following surgical resection is influenced by parameters detectable on the resection specimen or through a biopsy. The prognostic significance of HCC doubling time (DT) after surgery has never been investigated.
Methods
We evaluated 62 patients who underwent curative resection of a single HCC on cirrhosis; tumors were assessed before surgery on two subsequent occasions with the same imaging technique allowing the calculation of DT. The influence of tumor DT, clinical and pathological parameters on recurrence-rate and patients survival was assessed with uni- and multivariate analysis. Relationship between DT and pathological features was also analyzed.
Results
Three-year recurrence rate was 32.3% (20 patients): this was significantly higher in the presence of DT shorter than 100 days (58 versus 18% when equal to or longer; P=0.008), microvascular invasion (59 versus 17% when absent; P=0.008) or tumor undifferentiation (54 versus 25% when well/moderately differentiated; P=0.015). DT was the only independent predictor of recurrence (P=0.005). Patients survival was affected by Child-Pugh class only. DT was significantly shorter in tumors with microvascular invasion (P=0.007), undifferentiation (P=0.003) and high alpha-fetoprotein levels (P=0.011).
Conclusions
DT is easy to estimate and indicates the prognosis of single HCCs prior to liver resection.

Molecular and Cell Biology

Attenuated hepatic inflammation and fibrosis in angiotensin type 1a receptor deficient mice, 9 May 2005
Yang L, Bataller R, Dulyx J, Coffman TM, Ginès P, Rippe RA, Brenner DA
pages 317-323
Background/Aims
Pharmacological blockade of the renin–angiotensin system (RAS) attenuates liver fibrogenesis in rats. Here, we provide genetic evidence implicating angiotensin type 1 (AT1) receptors in liver fibrogenesis.
Methods
Wild type (WT) and AT1a knockout [AT1a (?/?)] mice were subjected to either sham operation or bile-duct ligation. Fibrosis was assessed by Sirius Red staining and hydroxyproline hepatic content. Fibrogenic and inflammatory cytokines were measured by ELISA.
Results
Bile duct ligation-induced elevation of serum liver enzymes was similar in WT and AT1a (?/?) mice. Bile duct ligated WT mice showed inflammatory changes and severe septal fibrosis. In contrast, AT1a (?/?) mice showed minor fibrotic lesions. Collagen accumulation was lower in AT1a (?/?) mice compared to WT mice. The increase in hepatic concentration of TGF?1 and pro-inflammatory cytokines was attenuated in AT1a (?/?) mice compared to WT mice. Immunohistochemistry analysis revealed decreased infiltration by inflammatory cells, lipid peroxidation products as well as decreased phosphorylation of c-Jun and p42/44 MAPK in AT1a (?/?) mice compared to AT1 (+/+) mice.
Conclusions
AT1 receptors play an important role in the development of fibrosis. Pharmacological blockade of AT1 receptors appears to be a promising approach to treat liver fibrosis.

Cholestasis and Autoimmune Liver Disease

Oxidative stress and apoptosis in fetal rat liver induced by maternal cholestasis. Protective effect of ursodeoxycholic acid, 3 May 2005
Perez MJ, Macias RIR, Duran C, Monte MJ, Gonzalez-Buitrago JM, Marin JJG
pages 324-332
Background/Aims
The sensitivity of fetal rat liver to maternal obstructive cholestasis during pregnancy (OCP), and the effect of ursodeoxycholic acid (UDCA) were investigated.
Methods
UDCA was administered (i.g. 0.6mg/kg b.wt./day) from day 14 to day 21 of pregnancy after maternal common bile duct ligation.
Results
Impairment in the activity of antioxidant enzymes, levels of total glutathione and GSH/GSSG ratio and the degrees of lipid peroxidation and protein carbonylation were similar in livers of OCP mothers and fetuses at term, despite hypercholanemia was milder in fetuses. Treatment of OCP rats with UDCA reduced maternal and fetal liver oxidative stress. Although maternal hypercholanemia was not corrected, fetal serum concentrations of major bile acids (except UDCA and ?-muricholic acid) were reduced. Fetal liver expression of key enzyme in bile acid synthesis, Cyp7a1, Cyp27 and Cyp8b1 was not affected by OCP or UDCA treatment. In OCP fetal livers, the relative expression of Bax-? and Bcl-2 and the activity of caspase-3, but not caspase-8, were increased. These changes were markedly reduced in fetuses of OCP animals treated with UDCA.
Conclusions
OCP induced moderate fetal hypercholanemia but marked liver oxidative stress and apoptosis that were partly prevented by treatment of pregnant rats with UDCA.

Genetic and Metabolic Liver Disease

Clinical, biochemical and morphological features of hepatocerebral syndrome with mitochondrial DNA depletion due to deoxyguanosine kinase deficiency, 31 May 2005
Labarthe F, Dobbelaere D, Devisme L, De Muret A, Jardel C, Taanman JW, Gottrand F, Lombès A
pages 333-341
Background/Aims
The aim of this study was to delineate the specific clinical, biological and liver morphological alterations of the hepatocerebral syndrome due to alterations in the deoxyguanosine kinase gene, a rare and severe form of mitochondrial DNA depletion syndrome.
Methods
We report seven cases from three unrelated families with the same mutation in the deoxyguanosine kinase gene.
Results
All the patients presented in the first weeks of life with hepatomegaly and progressive liver failure that led to death few months later. Major psychomotor delay and multidirectional nystagmus were reported shortly after onset of the disease. Severe hyperlactacidaemia was constant. Histological examination of the liver disclosed a multifocal injury of hepatocytes with irregular foamy steatosis, cholestasis, and fibrosis, associated with different degrees of hepatosiderosis and glycogen depletion. Liver respiratory chain activities were abnormal in all analysed patients and the amount of liver mitochondrial DNA was severely decreased. An identical homozygous 4bp GATT duplication was identified in the deoxyguanosine kinase gene of all the cases.
Conclusions
These patients, together with patients reported in the literature, permit to delineate the specific features of the hepatocerebral form of mitochondrial DNA depletion syndrome and to differentiate them from other causes of neonatal liver failure.

Copyright © 2001-2005  European Association for the Study of the Liver. All rights reserved.

BRITISH MEDICAL JOURNAL

Copyright © 2005 BMJ Publishing Group Ltd

NEW ENGLAND JOURNAL

The New England Journal of Medicine is owned, published, and copyrighted © 2005 Massachusetts Medical Society. All rights reserved.

LANCET

The Lancet, published, and copyrighted © 2005. All rights reserved.